RESUMEN
Nicotinamide phosphoribosyltransferase is a key metabolic enzyme that is a potential target for oncology. Utilizing publicly available crystal structures of NAMPT and in silico docking of our internal compound library, a NAMPT inhibitor, 1, obtained from a phenotypic screening effort was replaced with a more synthetically tractable scaffold. This compound then provided an excellent foundation for further optimization using crystallography driven structure based drug design. From this approach, two key motifs were identified, the (S,S) cyclopropyl carboxamide and the (S)-1-N-phenylethylamide that endowed compounds with excellent cell based potency. As exemplified by compound 27e such compounds could be useful tools to explore NAMPT biology in vivo.
Asunto(s)
Amidas/farmacología , Ciclopropanos/farmacología , Citocinas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Adenosina/análogos & derivados , Amidas/síntesis química , Amidas/química , Cristalografía por Rayos X , Ciclopropanos/síntesis química , Ciclopropanos/química , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Nicotinamida Fosforribosiltransferasa/metabolismo , Fenotipo , Relación Estructura-ActividadRESUMEN
Early-onset torsion dystonia is the most severe heritable form of dystonia, a human movement disorder that typically starts during a developmental window in early adolescence. Deletion in the DYT1 gene, encoding the torsinA protein, is responsible for this dominantly inherited disorder, which is non-degenerative and exhibits reduced penetrance among carriers. Here, we explore the hypothesis that deficits in torsinA function result in an increased vulnerability to stress associated with protein folding and processing in the endoplasmic reticulum (ER), where torsinA is located. Using an in vivo quantitative readout for the ER stress response, we evaluated the consequences of torsinA mutations in transgenic nematodes expressing variants of human torsinA. This analysis revealed that, normally, torsinA serves a protective function to maintain a homeostatic threshold against ER stress. Furthermore, we show that the buffering capacity of torsinA is greatly diminished by the DYT1-associated deletion or mutations that prevent its translocation to the ER, block ATPase activity, or increase the levels of torsinA in the nuclear envelope versus ER. Combinations of transgenic Caenorhabditis elegans designed to mimic clinically relevant genetic modifiers of disease susceptibility also exhibit a direct functional correlation to changes in the ER stress response. Furthermore, using mouse embryonic fibroblasts (MEFs) from torsinA knockout mice, we demonstrated that loss of endogenous torsinA results in enhanced sensitivity to ER stress. This study extends our understanding of molecular mechanisms underlying dystonia, and establishes a new functional paradigm to evaluate therapeutic strategies to compensate for reduced torsinA activity in the ER as a means to restore homeostatic balance and neuronal function.
Asunto(s)
Distonía/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Chaperonas Moleculares/metabolismo , Edad de Inicio , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Distonía/genética , Distonía/fisiopatología , Retículo Endoplásmico/genética , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Transporte de Proteínas , Estrés FisiológicoRESUMEN
Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinADeltaE). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinADeltaE when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinADeltaE mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinADeltaE message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinADeltaE acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinADeltaE to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.
Asunto(s)
Distonía Muscular Deformante/genética , Silenciador del Gen , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/genética , Secuencia de Bases , Técnicas de Cultivo de Célula , Células Cultivadas , Distonía Muscular Deformante/metabolismo , Distonía Muscular Deformante/patología , Fibroblastos/metabolismo , Genes Reporteros , Humanos , Luciferasas/metabolismo , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , TransfecciónRESUMEN
Small molecules that inhibit the metabolic enzyme NAMPT have emerged as potential therapeutics in oncology. As part of our effort in this area, we took a scaffold morphing approach and identified 3-pyridyl azetidine ureas as a potent NAMPT inhibiting motif. We explored the SAR of this series, including 5 and 6 amino pyridines, using a convergent synthetic strategy. This lead optimization campaign yielded multiple compounds with excellent in vitro potency and good ADME properties that culminated in compound 27.
RESUMEN
Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action.
Asunto(s)
Sistemas CRISPR-Cas , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Células Cultivadas , Inhibidores Enzimáticos/química , Eliminación de Gen , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Pruebas de Farmacogenómica/métodosRESUMEN
We have examined the distribution and ultrastructural localization of torsinA, the protein product of the TOR1A gene, in the normal adult human and Macaque brain. TorsinA immunoreactivity was visualized using a monoclonal antibody raised against a fusion protein encoding exon 4 of human torsinA. Western blot analysis of brain homogenates revealed a major species of about 39 kDa, consistent with the predicted size of glycosylated torsinA protein. By light microscopy, torsinA like-immunoreactivity was enriched in gray matter in all brain regions examined. Immunoreactivity was concentrated in the neuropil and immunopositive cell bodies were not observed. Structures particularly enriched in torsinA like-immunoreactivity included the cerebral cortex, the caudate-putamen, globus pallidus, the hippocampal formation, the thalamus, the substantia nigra and molecular cell layer of the cerebellar cortex. Cell bodies of pigmented dopamine neurons in the substantia nigra pars compacta were immunonegative. Biochemical fractionation of the human striata revealed a concentration of torsinA immunoreactivity in particulate fractions. Ultrastructural studies of the human and Macaque striata further revealed an association of torsinA immunostaining with small vesicles within axons and presynaptic terminals forming symmetric synapses. These ultrastructural studies are consistent with a pre-synaptic localization of torsinA protein in the adult striatum and are consistent with a role of torsinA in modulating striatal signaling, although the widespread localization of the protein suggests it probably also participates in signaling in other regions.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Chaperonas Moleculares , Terminales Presinápticos/metabolismo , Animales , Encéfalo/ultraestructura , Dendritas/metabolismo , Dendritas/ultraestructura , Humanos , Inmunohistoquímica , Macaca fascicularis , Microscopía Electrónica , Neostriado/metabolismo , Neostriado/ultraestructura , Terminales Presinápticos/ultraestructura , Proteínas Recombinantes de Fusión/inmunología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructuraRESUMEN
Translation initiation is a fine-tuned process that plays a critical role in tumorigenesis. The use of small molecules that modulate mRNA translation provides tool compounds to explore the mechanism of translational initiation and to further validate protein synthesis as a potential pharmaceutical target for cancer therapeutics. This report describes the development and use of a click beetle, dual luciferase cell-based assay multiplexed with a measure of compound toxicity using resazurin to evaluate the differential effect of natural products on cap-dependent or internal ribosome entry site (IRES)-mediated translation initiation and cell viability. This screen identified a series of cardiac glycosides as inhibitors of IRES-mediated translation using, in particular, the oncogene mRNA c-Myc IRES. Treatment of c-Myc-dependent cancer cells with these compounds showed a decrease in c-Myc protein associated with a significant modulation of cell viability. These findings suggest that inhibition of IRES-mediated translation initiation may be a strategy to inhibit c-Myc-driven tumorigenesis.
Asunto(s)
Glicósidos Cardíacos/análisis , Glicósidos Cardíacos/farmacología , Evaluación Preclínica de Medicamentos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ribosomas/metabolismo , Apoptosis/efectos de los fármacos , Secuencia de Bases , Bioensayo , Glicósidos Cardíacos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cimarina/química , Cimarina/farmacología , Daño del ADN , Genes Reporteros , Células HEK293 , Humanos , Concentración 50 Inhibidora , Inhibidores de la Síntesis de la Proteína/análisis , Inhibidores de la Síntesis de la Proteína/química , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Ribosomas/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Movement disorders represent a significant societal burden for which therapeutic options are limited and focused on treating disease symptomality. Early-onset torsion dystonia (EOTD) is one such disorder characterized by sustained and involuntary muscle contractions that frequently cause repetitive movements or abnormal postures. Transmitted in an autosomal dominant manner with reduced penetrance, EOTD is caused in most cases by the deletion of a glutamic acid (DeltaE) in the DYT1 (also known as TOR1A) gene product, torsinA. Although some patients respond well to anticholingerics, therapy is primarily limited to either neurosurgery or chemodenervation. As mutant torsinA (DeltaE) expression results in decreased torsinA function, therapeutic strategies directed toward enhancement of wild-type (WT) torsinA activity in patients who are heterozygous for mutant DYT1 may restore normal cellular functionality. Here, we report results from the first-ever screen for candidate small molecule therapeutics for EOTD, using multiple activity-based readouts for torsinA function in Caenorhabditis elegans, subsequent validation in human DYT1 patient fibroblasts, and behavioral rescue in a mouse model of DYT1 dystonia. We exploited the nematode to rapidly discern chemical effectors of torsinA and identified two classes of antibiotics, quinolones and aminopenicillins, which enhance WT torsinA activity in two separate in vivo assays. Representative molecules were assayed in EOTD patient fibroblasts for improvements in torsinA-dependent secretory function, which was improved significantly by ampicillin. Furthermore, a behavioral defect associated with an EOTD mouse knock-in model was also rescued following administration of ampicillin. These combined data indicate that specific small molecules that enhance torsinA activity represent a promising new approach toward therapeutic development for EOTD, and potentially for other diseases involving the processing of mutant proteins.
Asunto(s)
Modelos Animales de Enfermedad , Distonía Muscular Deformante/metabolismo , Chaperonas Moleculares/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Edad de Inicio , Ampicilina/química , Ampicilina/farmacología , Animales , Animales Modificados Genéticamente , Conducta Animal/efectos de los fármacos , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Evaluación Preclínica de Medicamentos , Distonía Muscular Deformante/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Ratones , Modelos Moleculares , Fármacos Neuroprotectores/farmacología , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/análisis , Relación Estructura-ActividadRESUMEN
A specific mutation (DeltaE) in torsinA underlies most cases of the dominantly inherited movement disorder, early-onset torsion dystonia (DYT1). TorsinA, a member of the AAA+ ATPase superfamily, is located within the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER). We investigated an association between torsinA and nesprin-3, which spans the outer nuclear membrane (ONM) of the NE and links it to vimentin via plectin in fibroblasts. Mouse nesprin-3alpha co-immunoprecipitated with torsinA and this involved the C-terminal region of torsinA and the KASH domain of nesprin-3alpha. This association with human nesprin-3 appeared to be stronger for torsinADeltaE than for torsinA. TorsinA also associated with the KASH domains of nesprin-1 and -2 (SYNE1 and 2), which link to actin. In the absence of torsinA, in knockout mouse embryonic fibroblasts (MEFs), nesprin-3alpha was localized predominantly in the ER. Enrichment of yellow fluorescent protein (YFP)-nesprin-3 in the ER was also seen in the fibroblasts of DYT1 patients, with formation of YFP-positive globular structures enriched in torsinA, vimentin and actin. TorsinA-null MEFs had normal NE structure, but nuclear polarization and cell migration were delayed in a wound-healing assay, as compared with wild-type MEFs. These studies support a role for torsinA in dynamic interactions between the KASH domains of nesprins and their protein partners in the lumen of the NE, with torsinA influencing the localization of nesprins and associated cytoskeletal elements and affecting their role in nuclear and cell movement.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Línea Celular , Movimiento Celular/genética , Proteínas del Citoesqueleto , Distonía Muscular Deformante/genética , Distonía Muscular Deformante/metabolismo , Embrión de Mamíferos/metabolismo , Retículo Endoplásmico/genética , Fibroblastos/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Chaperonas Moleculares/genética , Mutación , Proteínas del Tejido Nervioso/genética , Membrana Nuclear/genética , Proteínas Nucleares/genética , Plectina/genética , Plectina/metabolismo , Estructura Terciaria de Proteína/fisiología , Vimentina/genética , Vimentina/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
BACKGROUND: The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc). METHODOLOGY/PRINCIPLE FINDINGS: An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells. CONCLUSIONS/SIGNIFICANCE: The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.
Asunto(s)
Bioensayo , Retículo Endoplásmico/fisiología , Luciferasas/metabolismo , Vías Secretoras , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Copépodos/enzimología , Retículo Endoplásmico/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Riñón/citología , Riñón/metabolismo , Lentivirus/genética , Luciferasas/genética , Mediciones Luminiscentes , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
TorsinA is an AAA(+) protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope responsible for early onset torsion dystonia (DYT1). Most cases of this dominantly inherited movement disorder are caused by deletion of a glutamic acid in the carboxyl terminal region of torsinA. We used a sensitive reporter, Gaussia luciferase (Gluc) to evaluate the role of torsinA in processing proteins through the ER. In primary fibroblasts from controls and DYT1 patients most Gluc activity (95%) was released into the media and processed through the secretory pathway, as confirmed by inhibition with brefeldinA and nocodazole. Fusion of Gluc to a fluorescent protein revealed coalignment and fractionation with ER proteins and association of Gluc with torsinA. Notably, fibroblasts from DYT1 patients were found to secrete markedly less Gluc activity as compared with control fibroblasts. This decrease in processing of Gluc in DYT1 cells appear to arise, at least in part, from a loss of torsinA activity, because mouse embryonic fibroblasts lacking torsinA also had reduced secretion as compared with control cells. These studies demonstrate the exquisite sensitivity of this reporter system for quantitation of processing through the secretory pathway and support a role for torsinA as an ER chaperone protein.
Asunto(s)
Distonía/metabolismo , Distonía/patología , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Recuento de Células , Retículo Endoplásmico/metabolismo , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Inmunoprecipitación , Luciferasas/metabolismo , Ratones , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Solubilidad , Factores de TiempoRESUMEN
Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms-recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA-the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.
Asunto(s)
Encéfalo/metabolismo , Citoesqueleto/metabolismo , Chaperonas Moleculares/metabolismo , Mutación/fisiología , Neuritas/metabolismo , Vimentina/metabolismo , Actinas/metabolismo , Encéfalo/fisiopatología , Adhesión Celular/fisiología , Línea Celular , Línea Celular Tumoral , Forma de la Célula/fisiología , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/ultraestructura , Corriente Citoplasmática/fisiología , Distonía Muscular Deformante/metabolismo , Distonía Muscular Deformante/fisiopatología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Proteínas del Choque Térmico HSC70/metabolismo , Humanos , Cinesinas , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Neuritas/ultraestructura , Tubulina (Proteína)/metabolismoRESUMEN
Early onset dystonia is a movement disorder caused by loss of a glutamic acid residue (Glu(302/303)) in the carboxyl-terminal portion of the AAA+ protein, torsinA. We identified the light chain subunit (KLC1) of kinesin-I as an interacting partner for torsinA, with binding occurring between the tetratricopeptide repeat domain of KLC1 and the carboxyl-terminal region of torsinA. Coimmunoprecipitation analysis demonstrated that wild-type torsinA and kinesin-I form a complex in vivo. In cultured cortical neurons, both proteins co-localized along processes with enrichment at growth cones. Wild-type torsinA expressed in CAD cells co-localized with endogenous KLC1 at the distal end of processes, whereas mutant torsinA remained confined to the cell body. Subcellular fractionation of adult rat brain revealed torsinA and KLC associated with cofractionating membranes, and both proteins were co-immunoprecipitated after cross-linking cytoplasmically oriented proteins on isolated rat brain membranes. These studies suggest that wild-type torsinA undergoes anterograde transport along microtubules mediated by kinesin and may act as a molecular chaperone regulating kinesin activity and/or cargo binding.
Asunto(s)
Proteínas Portadoras/química , Proteínas Asociadas a Microtúbulos/química , Chaperonas Moleculares/química , Adenina/análogos & derivados , Adenina/metabolismo , Secuencia de Aminoácidos , Animales , Biotinilación , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Reactivos de Enlaces Cruzados/farmacología , Citoplasma/metabolismo , Distonía/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Cinesinas/química , Cinetina , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Neuronas/citología , Neuronas/metabolismo , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Estreptavidina/farmacología , Fracciones Subcelulares/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/metabolismoRESUMEN
The torsins comprise a four-member family of AAA+ chaperone proteins, including torsinA, torsinB, torp2A and torp3A in humans. Mutations in torsinA underlie early onset torsion dystonia, an autosomal dominant, neurologically based movement disorder. TorsinB is highly homologous to torsinA with its gene adjacent to that for torsinA on human chromosome 9q34. Antibodies have been generated which can distinguish torsinA and torsinB from each other, and from the torps in human and rodent cells. TorsinB (approximately MW 38 kDa), like torsinA ( approximately MW 37 kDa), is an N-glycosylated protein and both reside primarily in the endoplasmic reticulum (ER) and nuclear envelope in cultured cells. Immunoprecipitation studies in cultured cells and human brain tissue indicate that torsinA and torsinB are associated with each other in cells. Overexpression of both wild-type torsinB and mutant torsinA lead to enrichment of the protein in the nuclear envelope and formation of large cytoplasmic inclusions. We conclude that torsinB and torsinA are localized in overlapping cell compartments within the same protein complex, and thus may carry out related functions in vivo.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Chaperonas Moleculares/metabolismo , Neuroblastoma/metabolismo , Membrana Nuclear/metabolismo , Animales , Especificidad de Anticuerpos , Western Blotting , Encéfalo/citología , Química Encefálica , Proteínas Portadoras/análisis , Compartimento Celular/fisiología , Retículo Endoplásmico/metabolismo , Glicosilación , Humanos , Inmunohistoquímica , Riñón/citología , Riñón/metabolismo , Ratones , Chaperonas Moleculares/análisis , Neuroblastoma/patología , Membrana Nuclear/ultraestructura , Pruebas de PrecipitinaRESUMEN
TorsinA, a protein with homology to yeast heat shock protein104, has previously been demonstrated to colocalize with alpha-synuclein in Lewy bodies, the pathological hallmark of Parkinson's disease. Heat shock proteins are a family of chaperones that are both constitutively expressed and induced by stressors, and that serve essential functions for protein refolding and/or degradation. Here, we demonstrate that, like torsinA, specific molecular chaperone heat shock proteins colocalize with alpha-synuclein in Lewy bodies. In addition, using a cellular model of alpha-synuclein aggregation, we demonstrate that torsinA and specific heat shock protein molecular chaperones colocalize with alpha-synuclein immunopositive inclusions. Further, overexpression of torsinA and specific heat shock proteins suppress alpha-synuclein aggregation in this cellular model, whereas mutant torsinA has no effect. These data suggest that torsinA has chaperone-like activity and that the disease-associated GAG deletion mutant has a loss-of-function phenotype. Moreover, these data support a role for chaperone proteins, including torsinA and heat shock proteins, in cellular responses to neurodegenerative inclusions.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Alzheimer/patología , Proteínas Portadoras/genética , Línea Celular , Expresión Génica , Proteínas de Choque Térmico/genética , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Sustancias Macromoleculares , Chaperonas Moleculares/genética , Proteínas del Tejido Nervioso/genética , Unión Proteica/fisiología , Pliegue de Proteína , Sustancia Negra/patología , Sinucleínas , Transfección , alfa-SinucleínaRESUMEN
Familial, early onset, generalized torsion dystonia is the most common and severe primary dystonia. Most cases are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which is widely expressed in human brain and encodes the protein torsinA. This study compares neuropathology and torsinA expression in the normal human brain with that in dystonia cases with and without the GAG deletion. TorsinA-like protein was expressed in neuronal cytoplasm throughout the human brain, including cerebellum, substantia nigra, hippocampus, and neostriatum, with higher levels in specific neurons. This immunostaining pattern was not discernibly different in dystonia and normal brains in midbrain and neostriatal regions. However, nigral dopaminergic neurons appeared to be larger in both GAG-deletion and non-GAG-deletion dystonia brains compared to normal, and may be more closely spaced in GAG-deletion brains. Beyond these apparent changes in neuronal size and spacing in dystonia brains, there was no indication of neuron loss, inflammation, DNA strand breaks, or altered distribution of torsin-like immunoreactivity, supporting a functional rather than degenerative etiology of early onset torsion dystonia.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/patología , Proteínas Portadoras/metabolismo , Distonía Muscular Deformante/metabolismo , Distonía Muscular Deformante/patología , Eliminación de Gen , Chaperonas Moleculares , Neuronas/metabolismo , Neuronas/patología , Adolescente , Adulto , Anciano , Encéfalo/fisiopatología , Proteínas Portadoras/genética , Distonía Muscular Deformante/genética , Femenino , Genotipo , Humanos , Inmunohistoquímica , Lactante , Masculino , Persona de Mediana Edad , Mutación/genéticaRESUMEN
Most cases of early-onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the "ATPases associated with a variety of cellular activities" (AAA(+)) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor-induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress.