RESUMEN
Myelination was a major step in the evolution of the nervous system. Appearing first in jaw fish, myelination allows the fast and secure propagation of action potentials at a low energetic cost, and without exaggerated increase in axonal diameter. In the peripheral nervous system of mammals, myelination results from the tight interactions between Schwann cells and axons, leading to the formation of highly differentiated domains along the axon. The molecular determinants of these interactions are starting to be well identified. Their understanding provides a precise framework to interpret the defects, which occur in pathological circumstances. This review summarizes the present state of knowledge concerning axoglial interactions in peripheral nerves.
Asunto(s)
Axones/fisiología , Axones/ultraestructura , Células de Schwann/fisiología , Células de Schwann/ultraestructura , Animales , Axones/patología , Diferenciación Celular , Metabolismo Energético , Humanos , Vaina de Mielina/fisiología , Nervios Periféricos/fisiología , Nervios Periféricos/ultraestructura , Nódulos de Ranvier/fisiología , Nódulos de Ranvier/ultraestructura , Células de Schwann/patologíaAsunto(s)
Proteínas Portadoras/genética , Cromosomas Humanos Par 3/genética , Hibridación Fluorescente in Situ , Southern Blotting , Bandeo Cromosómico , ADN Complementario/genética , Humanos , Células Híbridas/metabolismo , Datos de Secuencia Molecular , Mapeo Físico de Cromosoma , Reacción en Cadena de la PolimerasaRESUMEN
The neurofibromatosis type 2 (NF2) tumor suppressor protein, known as schwannomin or merlin, is involved in linking membrane proteins to the cytoskeleton. Like the related ERM proteins, schwannomin has long been suspected of exhibiting a complex 3D organization caused by the association of different regions within the protein. Intramolecular interactions characterized to date are linking N-terminal sequences of the protein to C-terminal sequences. Here, we demonstrate, by a biochemical approach, the existence of a structured domain entirely contained within the N-terminal half of schwannomin. This structure, which is resistant to chymotryptic digestion, encompasses the FERM domain (residues 19-314), but excludes the 18 extreme N-terminal residues specific to schwannomin. The structure is disrupted by some, but not all, naturally occurring NF2 mutations. We investigated the significance of this structured domain in schwannomin cellular functions and found that normal schwannomin localization beneath the plasma membrane is directly dependent on proper folding of the N-terminal domain. In addition, folding of the N-terminal domain influences schwannomin interaction with actin through two novel actin-binding sites located in this region. These results suggest that loss of activity of several naturally occurring schwannomin mutants is due to disruption of the fold of the N-terminal domain, leading to loss of both membrane localization and actin association.
Asunto(s)
Actinas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Quimotripsina/farmacología , Células HeLa , Humanos , Proteínas de la Membrana/genética , Mutagénesis/fisiología , Neurofibromina 2 , Pliegue de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Hemizygosity for the NF2 gene in humans causes a syndromic susceptibility to schwannoma development. However, Nf2 hemizygous mice do not develop schwannomas but mainly osteosarcomas. In the tumors of both species, the second Nf2 allele is inactivated. We report that conditional homozygous Nf2 knockout mice with Cre-mediated excision of Nf2 exon 2 in Schwann cells showed characteristics of neurofibromatosis type 2. These included schwannomas, Schwann cell hyperplasia, cataract, and osseous metaplasia. Thus, the tumor suppressor function of Nf2, here revealed in murine Schwann cells, was concealed in hemizygous Nf2 mice because of insufficient rate of second allele inactivation in this cell compartment. The finding of this conserved function documents the relevance of the present approach to model the human disease.
Asunto(s)
Alelos , Genes de la Neurofibromatosis 2 , Mutación , Neurofibromatosis 2/genética , Animales , Secuencia de Bases , Cartilla de ADN , Exones , Eliminación de Gen , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Neurilemoma/genética , Regiones Promotoras GenéticasRESUMEN
The neurofibromatosis type 2 (NF2) protein, known as schwannomin or merlin, is a tumor suppressor involved in NF2-associated and sporadic schwannomas and meningiomas. It is closely related to the ezrin-radixin-moesin family members, implicated in linking membrane proteins to the cytoskeleton. The molecular mechanism allowing schwannomin to function as a tumor suppressor is unknown. In attempt to shed light on schwannomin function, we have identified a novel coiled-coil protein, SCHIP-1, that specifically associates with schwannomin in vitro and in vivo. Within its coiled-coil region, this protein is homologous to human FEZ proteins and the related Caenorhabditis elegans gene product UNC-76. Immunofluorescent staining of transiently transfected cells shows a partial colocalization of SCHIP-1 and schwannomin, beneath the cytoplasmic membrane. Surprisingly, immunoprecipitation assays reveal that in a cellular context, association with SCHIP-1 can be observed only with some naturally occurring mutants of schwannomin, or a schwannomin spliced isoform lacking exons 2 and 3, but not with the schwannomin isoform exhibiting growth-suppressive activity. Our observations suggest that SCHIP-1 interaction with schwannomin is regulated by conformational changes in schwannomin, possibly induced by posttranslational modifications, alternative splicing, or mutations.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neurofibromina 2 , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Empalme del ARN , Alineación de SecuenciaRESUMEN
Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. We tested whether this mechanism may also apply for the neurofibromatosis type-2 gene (NF2) which, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain showed high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein were normal. Our results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages.
Asunto(s)
Hiperplasia/etiología , Proteínas de la Membrana/fisiología , Neurilemoma/etiología , Células de Schwann , Animales , Humanos , Hiperplasia/patología , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Mutagénesis , Neurilemoma/patología , Neurofibromina 2 , Conejos , RatasRESUMEN
Germ-line mutations in the neurofibromatosis 2 (NF2) gene cause a susceptibility to the development of schwannoma and meningioma, 2 mostly benign tumors of neural crest origin. Bi-allelic inactivation of this gene has been observed in sporadic schwannomas and meningiomas. The NF2 gene may also be somatically inactivated in human malignant mesotheliomas (HMMs). Surprisingly, patients with an NF2 germ-line mutation have not been reported to be at an increased risk for this highly invasive tumor of mesodermal origin. To investigate in HMMs the silencing mechanism of the NF2 gene, we have analyzed its structure and expression in a series of 18 cell lines derived from HMMs. NF2 gene alterations were identified at a genomic level in 7 cell lines and were associated with a marked decrease in the concentration of the NF2 transcript. This decrease was also observed in 4 additional cell lines with no identified NF2 mutation. The 11 cell lines presented evidence suggesting deletion of one NF2 allele. None of these enabled the detection of normal or truncated forms of the NF2 protein by immunoprecipitational immunoblot analyses. In the 7 remaining cell lines, NF2 mRNA and NF2 protein were easily detectable. Among the latter, 4 lines were heterozygous for several chromosome 22 microsatellite loci, suggesting the presence of 2 NF2 alleles. Taken together, our data indicate that silencing of the NF2 gene is restricted to a subset of mesothelioma cell lines. The availability of established cell lines with different characterized NF2 status provides a powerful tool to explore the mechanism by which the NF2 protein exerts its tumor suppressive activity.
Asunto(s)
Genes Supresores de Tumor/genética , Pérdida de Heterocigocidad , Proteínas de la Membrana/genética , Mesotelioma/genética , Proteínas de Neoplasias/genética , Alelos , Electroforesis/métodos , Células HeLa , Humanos , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/análisis , Neurofibromina 2 , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Transiently expressed wild-type isoforms were both observed underneath the plasma membrane. At this location they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrated identical properties. Three different truncated schwannomins, that are prototypic for most naturally occurring NF2 mutations, were affected neither in their location nor in their cytochalasin D sensitivity. However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain had lost the submembraneous localization and tended to accumulate in perinuclear patches that were unaffected by cytochalasin D treatment. A similar behavior was observed when the N-terminal domain was entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Genes de la Neurofibromatosis 2 , Proteínas de la Membrana/metabolismo , Mutación Puntual , Citocalasina D/farmacología , Detergentes/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa , Humanos , Proteínas de la Membrana/química , Microscopía Inmunoelectrónica , Neurofibromina 2 , Nocodazol/farmacología , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Solubilidad , Relación Estructura-Actividad , Fracciones Subcelulares/química , TransfecciónRESUMEN
Transformation by rodent polyomaviruses is mediated primarily by middle T antigen, a membrane-bound protein that does not carry an intrinsic enzymatic activity but interacts and subverts the activity of cellular regulators of proliferation. The multiple protein partners of murine polyomavirus (Py) middle T antigen include the tyrosine kinases c-Src and, to a lesser extent, c-Fyn and c-Yes. By contrast, the hamster polyomavirus (HaPV) middle T antigen selectively activates the c-Fyn gene product. This difference may account for the contrasting tumor patterns induced by the two viruses. The sequences of the respective N-terminal and C-terminal functional domains of murine Py and HaPV middle T antigens are highly conserved whereas the intervening stretches are clearly divergent, leading to the speculation that this divergence may direct the specificity for tyrosine kinase activation. We have addressed this issue by constructing a chimera middle T antigen molecule carrying the N-terminal domain from HaPV (exon 1) in phase with the other two domains from murine Py (exon 2). The biological properties of this chimera molecule are indistinguishable from those of HaPV middle T antigen; it specifically activates p59c-Fyn and carries the transforming phenotype of the HaPV middle T antigen on rat fibroblasts.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Transformación Celular Viral/genética , Regulación Viral de la Expresión Génica , Infecciones por Polyomavirus/genética , Poliomavirus/genética , Proteínas Proto-Oncogénicas/genética , Infecciones Tumorales por Virus/genética , Animales , Secuencia de Bases , Cricetinae , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-fyn , Ratas , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Hamster polyomavirus (HaPV) causes lymphoma and leukemia when injected into newborn Syrian hamsters and achieves full transformation of rodent fibroblasts in vitro. It offers a comprehensive model to study at a molecular level the contributions of the viral oncogenes to neoplastic transformation in vitro and in the animal. We have investigated the ability of HaPV large T antigen to form a complex with the product of the retinoblastoma gene (pRb) in vitro. In this report, we demonstrate that HaPV large T antigen can indeed complex the pRb polypeptide. In order to investigate to what extent this interaction might contribute to tumor induction by the virus, we have introduced two different point mutations within the putative pRb-binding sequence of large T antigen, and as a preliminary to in vivo experiments we have studied their effects in vitro on some biological activities relevant to tumor induction. We show that the substitution (Glu-134-->Lys) obliterates pRb binding, suggesting that Glu-134 participates in the interaction between pRb and large T antigen, whereas the substitution (Glu-135-->Lys) has no effect. The Lys-134 mutation is strongly deleterious to the immortalization capacity of the viral genome, whereas the Lys-135 mutation has no effect. Neither of the two mutations affects the capacity of the viral genome to induce foci formation in the rat established cell line F111. These results indicate that the interaction between large T and pRb is required in the immortalization process but irrelevant to transformation. Both mutants show at least partial impairment of replication and productive cycle.
Asunto(s)
Antígenos Transformadores de Poliomavirus/metabolismo , Transformación Celular Neoplásica , Transformación Celular Viral , Poliomavirus/fisiología , Proteína de Retinoblastoma/metabolismo , Replicación Viral , Secuencia de Aminoácidos , Animales , Antígenos Transformadores de Poliomavirus/genética , Secuencia de Bases , Cricetinae , Mesocricetus , Datos de Secuencia Molecular , Mutación Puntual , Poliomavirus/genética , Poliomavirus/inmunología , Unión Proteica , Ratas , Relación Estructura-ActividadRESUMEN
A strategy involving polymerase chain reaction amplification of cDNAs was designed to study the expression of the hamster polyomavirus (HaPV) early region in HaPV-transformed rat fibroblasts, productively HaPV-infected cells, and HaPV-induced lymphoma. We identified three mRNAs resulting from alternative splicing of open reading frames leading to coding capacities for three polypeptides with molecular weights similar to those of the murine polyomavirus large T, middle T (MT), and small T (ST) antigens. The corresponding intronless cDNAs direct the in vitro synthesis of polypeptides with the expected electrophoretic mobilities. The biological activities carried by the HaPV early genes were assayed by transfection of appropriate cell systems. The fragment of genomic viral DNA that encodes the three early antigens contains all of the genetic information necessary for immortalization of primary rat embryo fibroblasts and transformation of F111 rat cells. The large T antigen is sufficient for immortalization, although the MT and ST antigens stimulate the growth and modify the phenotype of immortal cell lines. A stringent cooperative effect was observed in the transformation of F111 cells, which requires the simultaneous presence of the MT and ST antigens, as opposed to the transformation by murine polyomavirus, which can be carried out by the MT antigen alone.
Asunto(s)
Transformación Celular Viral/genética , Poliomavirus/genética , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Secuencia de Bases , Línea Celular , ADN Viral , Exones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Empalme del ARN , ARN Viral/metabolismo , Ratas , Ratas Endogámicas F344 , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Hamster polyomavirus (HaPV) is the causal agent of hair follicle epithelioma in hamsters belonging to a colony bred in Berlin-Buch. These tumors shed virus particles that are assembled in the keratinized layer of the epidermis. By contrast, HaPV induces lymphomas after inoculation into newborn hamsters from a distinct colony bred in Potsdam. These lymphoid tumors accumulate massive amounts of episomal viral genomes characterized by deletions that alter specifically the regulatory and the late coding sequences. Assuming that these alterations of the regulatory region may affect the transcription of the viral oncogenes in the tumor cells, the transcriptional activity of the wild-type and deleted early promoters have been studied in vitro in transient chloramphenicol acetyltransferase (CAT) expression assays. These assays performed in various cell types demonstrate that both versions of the HaPV early promoter carry a weak constitutive activity. Simultaneous expression of the HaPV early gene products leads to a strong stimulation of CAT activity with a concomitant activation of the replication of the plasmid constructs. The results obtained with origin-defective CAT vectors indicate that the replication contributes significantly to the stimulating effect of the early gene products. Indeed, transfection of massive amounts of CAT vectors that are unable to replicate can simulate the dosage effect of replication and also leads to measurable CAT activities. Under these conditions, the wild-type promoter is more active than the deleted version, indicating that sequences within the deletion carry a distinct stimulatory effect on transcription. This conclusion is supported by the observation that the lymphoma cells contain a low level of early transcripts, indicating that the deleted episomal viral templates accumulated in these tumors carry a weak transcriptional activity.
Asunto(s)
Regulación Viral de la Expresión Génica , Linfoma/veterinaria , Poliomavirus/genética , Animales , Secuencia de Bases , Cricetinae/microbiología , ADN Viral/genética , ADN Viral/ultraestructura , Genes Virales , Enlace de Hidrógeno , Linfoma/microbiología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Viral/genética , Transcripción Genética , Proteínas Estructurales Virales/genética , Replicación ViralRESUMEN
Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes.