RESUMEN
Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Canal de Sodio Activado por Voltaje NAV1.9/química , Canal de Sodio Activado por Voltaje NAV1.9/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Citosol/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico , Cinética , Canal de Sodio Activado por Voltaje NAV1.7/química , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Dominios Proteicos , Transporte de Proteínas , Relación Estructura-ActividadRESUMEN
POU1F1, a pituitary-specific POU-homeo domain transcription factor, plays an essential role in the specification of the somatotroph, lactotroph and thyrotroph lineages and in the activation of GH1, PRL and TSHß transcription. Individuals with mutations in POU1F1 present with combined deficiency of GH, PRL and TSH. Here, we identified a heterozygous missense mutation with evidence of pathogenicity, at the POU1F1 locus, in a large family in which an isolated growth hormone deficiency segregates as an autosomal dominant trait. The corresponding p.Pro76Leu mutation maps to a conserved site within the POU1F1 transactivation domain. Bandshift assays revealed that the mutation alters wild-type POU1F1 binding to cognate sites within the hGH-LCR and hGH1 promoter, but not to sites within the PRL promoter, and it selectively increases binding affinity to sites within the hGH-LCR. Co-immunoprecipitation studies reveal that this substitution enhances interactions of POU1F1 with three of its cofactors, PITX1, LHX3a and ELK1, and that residue 76 plays a critical role in these interactions. The insertion of the mutation at the mouse Pou1f1 locus results in a dramatic loss of protein expression despite normal mRNA concentrations. Mice heterozygous for the p.Pro76Leu mutation were phenotypically normal while homozygotes demonstrated a dwarf phenotype. Overall, this study unveils the involvement of POU1F1 in dominantly inherited isolated GH deficiency and demonstrates a significant impact of the Pro76Leu mutation on DNA-binding activities, alterations in transactivating functions and interactions with cofactors. Our data further highlight difficulties in modeling human genetic disorders in the mouse despite apparent conservation of gene expression pathways and physiologic functions.
Asunto(s)
Enanismo Hipofisario/genética , Mutación Missense , Carácter Cuantitativo Heredable , Factor de Transcripción Pit-1/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Enanismo Hipofisario/metabolismo , Enanismo Hipofisario/patología , Femenino , Regulación de la Expresión Génica , Genes Dominantes , Sitios Genéticos , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Heterocigoto , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Linaje , Hipófisis/metabolismo , Hipófisis/patología , Prolactina/genética , Prolactina/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal , Factor de Transcripción Pit-1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Renalase is a kidney-secreted catecholamines-degrading enzyme whose expression and activity are downregulated by increased dietary phosphate. A renalase knockout (KO) mouse model was used to explore the mechanisms mediating renalase's effect on phosphate excretion. Compared with wild-type (WT) mice maintained on a regular diet, KO mice show decreased serum PO4(-) (KO = 5.3 ± 0.2 vs. WT = 6.0 ± 0.1, n = 6; P < 0.04) and increased urinary PO4(-) excretion (urine PO4(-)/creatinine: KO = 7.7 ± 0.3 vs. WT = 6.1 ± 0.3, n = 6; P < 0.02). However, both WT and KO mice respond similarly to PO4(-) restriction by increasing renal COMT-1 activity and markedly decreasing PO4(-) excretion, which excludes an intrinsic renal defect in the KO. Renal sodium-phosphate cotransporter Npt2a, sodium proton exchanger NHE3 expression, and MAO-A and B activity did not differ between WT and KO. Only catechol-O-methyl transferase (COMT) expression and activity were significantly increased in KO mice. Despite that, urinary dopamine increased by twofold, whereas urinary l-DOPA excretion decreased by twofold in the KO mouse, indicating an upregulation of renal dopamine (DA) synthesis. These data indicate that renalase deficiency is associated with increased renal DA synthesis, stimulated PO4(-) excretion, and moderately severe hypophosphatemia. The signal to increase renal DA synthesis is strong since it overcomes a compensatory increase in COMT activity.
Asunto(s)
Dopamina/metabolismo , Levodopa/orina , Monoaminooxidasa/fisiología , Fosfatos/orina , Animales , Catecol O-Metiltransferasa/metabolismo , Creatinina/sangre , Dopamina/orina , Hipofosfatemia/etiología , Riñón/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Amyloid light-chain (AL) amyloidosis is the second most common form of systemic amyloidosis which is characterized by a high level of mortality and no effective treatment to remove fibril deposition. This disorder is caused by malfunctioning of B-cells resulting in production of abnormal protein fibrils composed of immunoglobulin light chain fragments that tend to deposit on various organs and tissues. AL amyloidosis is set apart from other forms of amyloidosis in that no specific sequences have been identified in the immunoglobulin light chains that are amyloid fibril formation causative and patient specific. This unusual feature hinders the therapeutic progress and requires either direct access to patient samples (which is not always possible) or a source of in vitro produced fibrils. While isolated reports of successful AL amyloid fibril formation from various patient-specific protein sequences can be found in literature, no systematic research on this topic was performed since 1999. In the present study we have developed a generalized approach to in vitro fibril production from various types of previously reported [[1], [2], [3]] amyloidogenic immunoglobulin light chains and their fragments. We describe the procedure from selection and generation of starting material, through finding of optimal assay conditions, to applying a panel of methods to confirm successful fibril formation. Procedure details are discussed in the light of the most recent findings and theories on amyloid fibril formation. The reported protocol produces high quality AL amyloid fibrils that can subsequently be used in the development of the much-needed amyloid-targeting diagnostic and therapeutic approaches.
RESUMEN
The hGH cluster contains a single human pituitary growth hormone gene (hGH-N) and four placenta-specific paralogs. Activation of the cluster in both tissues depends on 5' remote regulatory elements. The pituitary-specific locus control elements DNase I-hypersensitive site I (HSI) and HSII, located 14.5 kb 5' of the cluster (position -14.5), establish a continuous domain of histone acetylation that extends to and activates hGH-N in the pituitary gland. In contrast, histone modifications in placental chromatin are restricted to the more 5'-remote HSV-HSIII region (kb -28 to -32) and to the placentally expressed genes in the cluster, with minimal modification between these two regions. These data predict distinct modes of hGH cluster gene activation in the pituitary and placenta. Here we used cell culture models to track structural changes at the hGH locus through placental-gene activation. The data revealed that this process was initiated in primary cytotrophoblasts by histone H3K4 di- and trimethylation and H4 acetylation restricted to HSV and to the individual placental-gene repeat (PGR) units within the cluster. Later stages of transcriptional induction were accompanied by enhancement and extension of these modifications and by robust H3 acetylation at HSV, at HSIII, and throughout the placental-gene regions. These data suggested that elements restricted to HSIII-HSV regions and each individual PGR might be sufficient for activation of the hCS genes. This model was tested by comparing hCS transgene expression in the placentas of mouse embryos carrying a full hGH cluster to that in placentas in which the HSIII-HSV region was directly linked to the individual hCS-A PGR unit. The findings indicate that the HSIII-HSV region and the PGR units, although targeted for initial chromatin structural modifications, are insufficient to activate gene expression and that this process is dependent on additional, as-yet-unidentified chromatin determinants.
Asunto(s)
Diferenciación Celular , Epigénesis Genética , Hormona de Crecimiento Humana/genética , Placenta/metabolismo , Trofoblastos/fisiología , Células Cultivadas , Inmunoprecipitación de Cromatina , Femenino , Hormona de Crecimiento Humana/metabolismo , Humanos , Modelos Genéticos , Familia de Multigenes , Embarazo , Tercer Trimestre del Embarazo , Trofoblastos/citologíaRESUMEN
X-ray repair cross complementing protein 1 (XRCC1) plays an important role in base excision DNA repair (BER) as a scaffolding protein for BER enzymes. BER is one of the basic DNA repair pathways repairing greater than 20,000 endogenous lesions per cell per day. Proper functioning of XRCC1, one of the most important players in BER, was suggested to be indispensable for effective DNA repair. Despite accumulating evidence of an important role that XRCC1 plays in maintaining genomic stability, the relationship between one of its most predominant variants, R280H (rs25489), and cancer prevalence remains ambiguous. In the current study we functionally characterized the effect of the R280H variant expression on immortal non-transformed mouse mammary epithelial C127 and human breast epithelial MCF10A cells. We found that expression of R280H results in increased focus formation in mouse C127 cells and induces cellular transformation in human MCF10A cells. Cells expressing R280H showed significantly increased levels of chromosomal aberrations and accumulate double strand breaks in the G1 cell cycle phase. Our results confirm a possible link between R280H and genomic instability and suggest that individuals carrying this mutation may be at increased risk of cancer development.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Animales , Neoplasias de la Mama/genética , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Clonación Molecular , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Fase G1 , Variación Genética , Humanos , Ratones , Factores de Riesgo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
We report the establishment of three distinct pituitary-derived murine cell lines generated by targeted T-antigen-induced transformation. The Pit1/0 line expresses pituitary-specific transcription factor-1 (Pit-1) but lacks expression of GH, prolactin (Prl), or TSH, and the Pit1/Prl line is selectively positive for Pit-1 and Prl. The third line, Pit1/Triple, expresses Pit-1 and all three of the Pit-1-dependent hormones: GH, Prl, and TSHß/glycoprotein hormone α-subunit. The three corresponding transformation events appear to have captured pituitary cells representing: 1) an initial step in the Pit-1(+) lineage, 2) a cell line that corresponds to the differentiated lactotrope, and 3) a novel tri-hormone intermediate that may represent a pivotal step in Pit-1(+) cell lineage differentiation. The documented dependence of the tri-hormone expression in the Pit-1/Triple line on Pit-1 activity supports its potential role in the pathway of pituitary cell differentiation. The presence of a 123-kb human transgene encompassing the hGH locus (hGH/bacterial artificial chromosome) in two of these lines, Pit1/0 and Pit1/Prl, further expands their potential utility to the analysis of gene activation within the hGH gene cluster.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Línea Celular Transformada/citología , Línea Celular Transformada/patología , Linaje de la Célula , Transformación Celular Neoplásica/patología , Hipófisis/patología , Factor de Transcripción Pit-1/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Transgénicos , Modelos Biológicos , Hipófisis/metabolismo , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Pit-1/genética , Transgenes/genéticaRESUMEN
We used a beta-amyloid precursor protein (APP) transgenic (Tg) mouse model that displays some of the typical Alzheimer-associated pathological features to study the brain proteoma associated with amyloid plaque deposition. Two groups (male and female) of 14-month-old Tg mice were compared with their wild type littermates. We used differential 2D electrophoresis coupled with mass spectrometry to generate one of the first complete image of changes in brain protein expression occurring in this well-recognized model of Alzheimer's disease (AD). We identified 15 different proteins, which are significantly regulated in this pathology (p<0.05, > or =1.5-fold variation in expression comparing with the wild type samples). These comprise a number of proteins that were already known to be implicated in AD and neurodegeneration, as well as several proteins which relationship with AD had not been shown before. Identified proteins were grouped according to their biological key pathways. Results obtained are discussed in view of existing bibliographic data on human AD transcriptoma and proteoma.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Electroforesis en Gel Bidimensional/métodos , Proteómica/métodos , Precursor de Proteína beta-Amiloide/genética , Animales , Apolipoproteínas E/metabolismo , Western Blotting/métodos , Encéfalo/patología , Diagnóstico por Imagen/métodos , Modelos Animales de Enfermedad , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.