RESUMEN
Transglutaminases are calcium-dependent enzymes that catalyze the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific glutamine and lysine residues. Some transglutaminase isoforms are present in the brain and are thought to participate in the protein aggregation characteristic of neurological diseases such as Huntington, Alzheimer's and Parkinson's disease. We have developed a functional proteomics strategy in which biotinylated amine-donor and amine-acceptor probes were used to identify the transglutaminase substrates present in brain. Bioinformatics analyses revealed that most of the 166 brain substrates identified interacted with huntingtin, the amyloid precursor protein or α-synuclein and that neurological disease was the most significant canonical pathway associated with the substrates. The physiological relevance of the substrates identified by mass spectrometry was confirmed by the fact that three of them (actin, ß-tubulin and a neurofilament subunit) were polymerized in neuronal cells when cytosolic calcium concentration was raised. We also showed by in-situ immunolabeling that some of the substrates were part of the protein aggregates found in neurological diseases. These results strongly support the idea that the crosslinking activity of brain transglutaminase participates in the formation of the protein aggregates found in diseases of the central nervous system.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Enfermedad de Huntington/metabolismo , Proteoma , Transglutaminasas/metabolismo , Adolescente , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Encéfalo/patología , Línea Celular Tumoral , Femenino , Humanos , Enfermedad de Huntington/patología , Masculino , Persona de Mediana Edad , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Placa Amiloide/metabolismo , Placa Amiloide/patología , ProteómicaRESUMEN
R6/2 mice contain an N-terminal fragment of human huntingtin with an expanded polyQ and develop a neurological disease resembling Huntington disease. Although the brain of R6/2 mice contains numerous inclusions, there is very little neuronal death. In that respect, R6/2 mice differ from patients with Huntington disease whose striatum and cerebral cortex develop inclusions associated with extensive neuronal loss. We have previously demonstrated using synchrotron-based infrared microspectroscopy that the striatum and the cortex of patients with Huntington disease contained inclusions specifically enriched in amyloid ß-sheets. We had concluded that the presence of an amyloid motif conferred toxicity to the inclusions. We demonstrate here by synchrotron based infrared microspectroscopy in transmission and attenuated total reflectance mode that the inclusions of R6/2 mice possess no detectable amyloid and are composed of proteins whose structure is not distinguishable from that of the surrounding soluble proteins. The difference in structure between the inclusions of patients affected by Huntington disease and those of R6/2 mice might explain why the former but not the latter cause neuronal death.
Asunto(s)
Amiloide/metabolismo , Encéfalo/metabolismo , Proteína Huntingtina/genética , Amiloide/química , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Ratones , Ratones Transgénicos , Microscopía , Péptidos/química , Péptidos/metabolismo , Análisis de Componente Principal , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Absence of mitosis and meiosis are distinguishing properties of male germ cells during late fetal and early neonatal periods. Repressors of male germ cell meiosis have been identified, but mitotic repressors are largely unknown, and no protein repressing both meiosis and mitosis is known. We demonstrate here that the zinc-finger protein BNC2 is present in male but not in female germ cells. In testis, BNC2 exists as several spliced isoforms and presumably binds to DNA. Within the male germ cell lineage, BNC2 is restricted to prospermatogonia and undifferentiated spermatogonia. Fetal prospermatogonia that lack BNC2 multiply excessively on embryonic day (E)14.5 and reenter the cell cycle prematurely. Mutant prospermatogonia also engage in abnormal meiosis; on E17.5, Bnc2(-/-) prospermatogonia start synthesizing the synaptonemal protein SYCP3, and by the time of birth, many Bnc2(-/-) prospermatogonia have accumulated large amounts of nonfilamentous SYCP3, thus appearing to be blocked at leptonema. Bnc2(-/-) prospermatogonia do not undergo proper male differentiation, as they lack almost all the mRNA for the male-specific methylation protein DNMT3L and have increased levels of mRNAs that encode meiotic proteins, including STRA8. Bnc2(-/-) prospermatogonia can produce spermatogonia, but these enter meiosis prematurely and undergo massive apoptotic death during meiotic prophase. This study identifies BNC2 as a major regulator of male germ stem cells, which is required for repression of meiosis and mitosis in prospermatogonia, and for meiosis progression during spermatogenesis. In view of the extreme evolutionary conservation of BNC2, the findings described here are likely to apply to many species.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Meiosis/fisiología , Mitosis/fisiología , Espermatogénesis/fisiología , Espermatogonias/fisiología , Animales , Proteínas de Ciclo Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Inmunohistoquímica , Masculino , Meiosis/genética , Ratones , Ratones Noqueados , Mitosis/genética , Proteínas Nucleares/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Espermatogénesis/genética , Espermatogonias/metabolismoRESUMEN
Huntington's disease is caused by a polyglutamine expansion in huntingtin. Affected brain regions contain characteristic aggregates of the misfolded expanded protein. Studies in cells and animals show that aggregates are polymorphic and that the secondary structure of the aggregates is likely to condition their cytotoxicity. Therefore knowing the structure of aggregates is important as neurotoxic secondary structures may be specifically targeted during the search for prophylactic or therapeutic drugs. The structure of aggregates in the brain of patients is still unknown. Using synchrotron based infrared microspectroscopy we demonstrate that the brains of patients with Huntington disease contain putative oligomers and various kinds of microscopic aggregates (inclusions) that can be distinguished by their differential absorbance at 1627 cm(-1) (amyloid ß sheets) and 1639 cm(-1) (ß sheets/unordered). We also describe the parallel/antiparallel organization of the ß strands. As the inclusions enriched in both ß sheets and ß sheets/unordered structures are characteristic of severely affected brain regions, we conclude that this kind of amyloid inclusions is likely to be particularly toxic to neurons.
Asunto(s)
Amiloide/análisis , Encéfalo/patología , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/análisis , Espectrofotometría Infrarroja/instrumentación , Sincrotrones/instrumentación , Humanos , Proteína Huntingtina , Estructura Secundaria de ProteínaRESUMEN
Basonuclin 2 is a recently discovered zinc finger protein of unknown function. Its paralog, basonuclin 1, is associated with the ability of keratinocytes to multiply. The basonuclin zinc fingers are closely related to those of the Drosophila proteins disco and discorelated, but the relation between disco proteins and basonuclins has remained elusive because the function of the disco proteins in larval head development seems to have no relation to that of basonuclin 1 and because the amino acid sequence of disco, apart from the zinc fingers, also has no similarity to that of the basonuclins. We have generated mice lacking basonuclin 2. These mice die within 24 h of birth with a cleft palate and abnormalities of craniofacial bones and tongue. In the embryonic head, expression of the basonuclin 2 gene is restricted to mesenchymal cells in the palate, at the periphery of the tongue, and in the mesenchymal sheaths that surround the brain and the osteocartilagineous structures. In late embryos, the rate of multiplication of these mesenchymal cells is greatly diminished. Therefore, basonuclin 2 is essential for the multiplication of craniofacial mesenchymal cells during embryogenesis. Non-Drosophila insect databases available since 2008 reveal that the basonuclins and the disco proteins share much more extensive sequence and gene structure similarity than noted when only Drosophila sequences were examined. We conclude that basonuclin 2 is both structurally and functionally the vertebrate ortholog of the disco proteins. We also note the possibility that some human craniofacial abnormalities are due to a lack of basonuclin 2.
Asunto(s)
Proliferación Celular , Proteínas de Unión al ADN/fisiología , Células Madre Mesenquimatosas/citología , Animales , Animales Recién Nacidos , Northern Blotting , Línea Celular , Fisura del Paladar/embriología , Fisura del Paladar/genética , Anomalías Craneofaciales/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Regulación del Desarrollo de la Expresión Génica , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cráneo/citología , Cráneo/embriología , Cráneo/metabolismo , Lengua/anomalías , Lengua/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
Basonuclins 1 and 2 are nuclear proteins specific to vertebrates. They have recently been shown to be the orthologs of the DISCO proteins of insects. All of these proteins have an essential function in embryonic development. It is likely that they are transcription factors with multiple gene targets, some of which are transcribed by DNA polymerase I, and others by polymerase II. It remains to be found which targets are common to the two basonuclins and even to the DISCO proteins, and why certain cell types possess either basonuclin 1 or basonuclin 2, while others possess both. The implication of the basonuclins in several human diseases adds to their interest as regulatory proteins.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/fisiología , Insectos/embriología , Factores de Transcripción/fisiología , Vertebrados/embriología , Secuencia de Aminoácidos , Animales , División Celular/genética , División Celular/fisiología , Secuencia de Consenso , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Humanos , Proteínas de Insectos/genética , Insectos/genética , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genética , Vertebrados/genética , Dedos de Zinc/genética , Dedos de Zinc/fisiologíaRESUMEN
The cross-linked (cornified) envelope is a characteristic product of terminal differentiation in the keratinocyte of the epidermis and related epithelia. This envelope contains many proteins of which involucrin was the first to be discovered and shown to become cross-linked by a cellular transglutaminase. Involucrin has evolved greatly in placental mammals, but retains the glutamine repeats that make it a good substrate for the transglutaminase. Until recently, it has been impossible to detect involucrin outside the placental mammals, but analysis of the GenBank and Ensembl databases that have become available since 2006 reveals the existence of involucrin in marsupials and birds. We describe here the properties of these involucrins and the ancient history of their evolution.
Asunto(s)
Epidermis/metabolismo , Epitelio/metabolismo , Evolución Molecular , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Monodelphis/genética , Monodelphis/metabolismo , Alineación de SecuenciaRESUMEN
Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that-in many aspects-recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that-unlike two- or three-lens light-sheet microscopes-it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petrán spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.
RESUMEN
Protein aggregates are characteristic of a number of diseases of the central nervous system such as diseases of polyQ expansion. Covalent bonds formed by the action of transglutaminase are thought to participate in the stabilization of these aggregates. Transglutaminase catalyzes the formation of cross-links between the side chains of glutaminyl and lysyl residues of polypeptides. Identification of the isodipeptide N(epsilon)-(gamma-glutamyl) lysine (iEK) in terminal proteolytic digests of neuronal aggregates would demonstrate participation of transglutaminase in neurological diseases. In order to identify and quantify the iEK present in the brain of patients with neurological disease, a method combining liquid chromatography and multistep mass spectrometry was developed. Because isobaric peptides of iEK could be present in the digest of aggregated proteins, the choice of fragment diagnostic ions was crucial. These ions were identified by mass spectrometry on sodiated iEK, which was derivatized on the carboxylic functions and terminal amines in order to improve sensitivity. Deuterated molecules as well as (13)C(6)- and (15)N(2)-isotopomers were used to derive filiations in the multistep fragmentations. The main fragmentation patterns have been identified, so that two ions (m/z 396 [MH - 56-42 u](+) and 350 [MH - 56-88 u](+)) are shown to be adequate markers for quantitation experiments. In order to gain a better understanding of the fragmentation processes, detailed quantum chemical calculations have been performed at levels which are expected to provide good accuracy. A thorough study has been carried out with a reduced model in which only the 'active' part of the molecule is retained. This allowed obtaining full mechanistic details on the pathways leading to a number of observed fragments. In particular, it has been shown that losses of 87 and 88 u from A(+) = [MH - 56 u](+) are competitive. Computations on the entire derivatized isodipeptide have been used to validate the use of the smaller model in order to obtain reliable energetics and mechanisms.
Asunto(s)
Biomarcadores/química , Dipéptidos/química , Espectrometría de Masas/métodos , Modelos Químicos , Enfermedades del Sistema Nervioso/metabolismo , Biomarcadores/metabolismo , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , Dipéptidos/metabolismo , Humanos , Enfermedades del Sistema Nervioso/diagnóstico , Péptidos/química , Péptidos/metabolismo , Transglutaminasas/metabolismoRESUMEN
Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by the specific transcription activators IFN regulatory factor 3 (IRF3) and IRF-7 and the homeoprotein transcription repressor Pitx1. We now show that repression by Pitx1 does not appear to be due to the recruitment of histone deacetylases. On the other hand, Pitx1 inhibits the IRF3 and IRF7 transcriptional activity of the IFN-A11 and IFN-A5 promoters and interacts physically with IRF3 and IRF7. Pitx1 trans-repression activity maps to specific C-terminal domains, and the Pitx1 homeodomain is involved in physical interaction with IRF3 or IRF7. IRF3 is able to bind to the antisilencer region of the IFN-A4 promoter, which overrides the repressive activity of Pitx1. These results indicate that interaction between the Pitx1 homeodomain and IRF3 or IRF7 and the ability of the Pitx1 C-terminal repressor domains to block IFN-A11 and IFN-A5 but not IFN-A4 promoter activities may contribute to our understanding of the complex differential transcriptional activation, repression, and antirepression of the IFN-A genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Interferón-alfa/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Células HeLa , Histona Desacetilasas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Factor 3 Regulador del Interferón , Factor 7 Regulador del Interferón , Factores de Transcripción Paired Box , Proteínas Represoras/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The involucrin gene encodes a protein of terminally differentiated keratinocytes. Its segment of repeats, which represents up to 80% of the coding region, is highly polymorphic in mouse strains derived from wild progenitors. Polymorphism includes nucleotide substitutions, but is most strikingly due to the recent addition of a variable number of repeats at a precise location within the segment of repeats. Each mouse taxon examined showed consistent and distinctive patterns of evolution of its variable region: very rapid changes in most M. m. domesticus alleles, slow changes in M. m. musculus, and complete arrest in M. spretus. We conclude that changes in the variable region are controlled by the genetic background. One of the M. m. domesticus alleles (DIK-L), which is of M. m. musculus origin, has undergone a recent repeat duplication typical of M. m. domesticus. This suggests that the genetic background controls repeat duplications through trans-acting factors. Because the repeat pattern differs in closely related murine taxa, involucrin reveals with greater sensitivity than random nucleotide substitutions the evolutionary relations of the mouse and probably of all murids.
Asunto(s)
Precursores de Proteínas/genética , Secuencias Repetitivas de Ácidos Nucleicos , Alelos , Animales , Secuencia de Bases , Southern Blotting , Diferenciación Celular , Linaje de la Célula , Evolución Molecular , Queratinocitos/metabolismo , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
BNC2 is an extremely conserved zinc finger protein with important functions in the development of craniofacial bones and male germ cells. Because disruption of the Bnc2 gene in mice causes neonatal lethality, the function of the protein in adult animals has not been studied. Until now BNC2 was considered to have a wider tissue distribution than its paralog, BNC1, but the precise cell types expressing Bnc2 are largely unknown. We identify here the cell types containing BNC2 in the mouse and we show the unexpected presence of BNC1 in many BNC2-containing cells. BNC1 and BNC2 are colocalized in male and female germ cells, ovarian epithelial cells, sensory neurons, hair follicle keratinocytes and connective cells of organ capsules. In many cell lineages, the two basonuclins appear and disappear synchronously. Within the male germ cell lineage, BNC1 and BNC2 are found in prospermatogonia and undifferentiated spermatogonia, and disappear abruptly from differentiating spermatogonia. During oogenesis, the two basonuclins accumulate specifically in maturing oocytes. During the development of hair follicles, BNC1 and BNC2 concentrate in the primary hair germs. As follicle morphogenesis proceeds, cells possessing BNC1 and BNC2 invade the dermis and surround the papilla. During anagen, BNC1 and BNC2 are largely restricted to the basal layer of the outer root sheath and the matrix. During catagen, the compartment of cells possessing BNC1 and BNC2 regresses, and in telogen, the two basonuclins are confined to the secondary hair germ. During the next anagen, the BNC1/BNC2-containing cell population regenerates the hair follicle. By examining Bnc2(-/-) mice that have escaped the neonatal lethality usually associated with lack of BNC2, we demonstrate that BNC2 possesses important functions in many of the cell types where it resides. Hair follicles of postnatal Bnc2(-/-) mice do not fully develop during the first cycle and thereafter remain blocked in telogen. It is concluded that the presence of BNC2 in the secondary hair germ is required to regenerate the transient segment of the follicle. Postnatal Bnc2(-/-) mice also show severe dwarfism, defects in oogenesis and alterations of palatal rugae. Although the two basonuclins possess very similar zinc fingers and are largely coexpressed, BNC1 cannot substitute for BNC2. This is shown incontrovertibly in knockin mice expressing Bnc1 instead of Bnc2 as these mice invariably die at birth with craniofacial abnormalities undistinguishable from those of Bnc2(-/-) mice. The function of the basonuclins in the secondary hair germ is of particular interest.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Animales , Linaje de la Célula/fisiología , Dermis/metabolismo , Células Epiteliales/metabolismo , Femenino , Folículo Piloso/metabolismo , Queratinocitos/metabolismo , Masculino , Ratones , Oocitos/metabolismo , Oogénesis/fisiología , Células Receptoras Sensoriales/metabolismo , Espermatogonias/metabolismo , Dedos de Zinc/fisiologíaRESUMEN
Alzheimer's disease, Parkinson's disease and diseases of expanded polyglutamine are associated with insoluble protein aggregates and neuronal death. A role for transglutaminase in the stabilization of these aggregates has been proposed. Diseases of polyglutamine expansion have been the most thoroughly investigated and a large body of studies supports the causative role of transglutaminase in aggregation of expanded polyglutamine. However none of the evidence is conclusive. Indisputable evidence of cross-linking by transglutaminase will be required in order to provide firm support for therapeutic measures based on the role of transglutaminase.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Péptidos/fisiología , Transglutaminasas/metabolismo , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Humanos , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/metabolismo , Enfermedades Neurodegenerativas/enzimología , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/metabolismo , Péptidos/metabolismoRESUMEN
Huntington disease is a dominantly inherited disease of the central nervous system. The mutational expansion of polyglutamine beyond a critical length produces a toxic gain of function in huntingtin and results in neuronal death. In the course of the disease, expanded huntingtin is proteolyzed, becomes abnormally folded, and accumulates in oligomers, fibrils, and microscopic inclusions. The aggregated forms of the expanded protein are structurally diverse. Structural heterogeneity may explain why polyglutamine-containing aggregates could paradoxically be either toxic or neuroprotective. When defined, the toxic structures could then specifically be targeted by prophylactic or therapeutic drugs aimed at inhibiting polyglutamine aggregation.
Asunto(s)
Amiloide/metabolismo , Enfermedad de Huntington/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Agregación Patológica de Proteínas/metabolismo , Amiloide/química , Animales , Animales Modificados Genéticamente , Birrefringencia , Encéfalo/ultraestructura , Epítopos/química , Epítopos/inmunología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/patología , Cuerpos de Inclusión , Ratones , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Péptido Hidrolasas/metabolismo , Péptidos/inmunología , Polimerizacion , Agregado de Proteínas/fisiología , Pliegue de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas , Ratas , Solubilidad , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
The presence of an expanded polyglutamine produces a toxic gain of function in huntingtin. Protein aggregation resulting from this gain of function is likely to be the cause of neuronal death. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation and covalent bonding by transglutaminase-catalyzed cross-linking. In cell culture models of Huntington's disease, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Nothing is known about the nature of the bonds that stabilize the aggregates in the brain of patients with Huntington's disease. It seems that the nature of the bond stabilizing the aggregates is one of the most important questions, as the answer would condition the therapeutic approach to Huntington's disease.
Asunto(s)
Enfermedad de Huntington/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Pliegue de Proteína , Animales , Muerte Celular/fisiología , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Citoplasma/metabolismo , Humanos , Proteína Huntingtina , Enfermedad de Huntington/etiología , Cuerpos de Inclusión/metabolismo , Proteínas del Tejido Nervioso/química , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/química , Fragmentos de Péptidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Transglutaminasas/metabolismoRESUMEN
Huntington disease and other diseases of polyglutamine expansion are each caused by a different protein bearing an excessively long polyglutamine sequence and are associated with neuronal death. Although these diseases affect largely different brain regions, they all share a number of characteristics, and, therefore, are likely to possess a common mechanism. In all of the diseases, the causative protein is proteolyzed, becomes abnormally folded and accumulates in oligomers and larger aggregates. The aggregated and possibly the monomeric expanded polyglutamine are likely to play a critical role in the pathogenesis and there is increasing evidence that the secondary structure of the protein influences its toxicity. We describe here, with special attention to huntingtin, the mechanisms of polyglutamine aggregation and the modulation of aggregation by the sequences flanking the polyglutamine. We give a comprehensive picture of the characteristics of monomeric and aggregated polyglutamine, including morphology, composition, seeding ability, secondary structure, and toxicity. The structural heterogeneity of aggregated polyglutamine may explain why polyglutamine-containing aggregates could paradoxically be either toxic or neuroprotective.
RESUMEN
Basonuclin 1 and the recently discovered basonuclin 2 are vertebrate proteins with multiple paired C(2)H(2) zinc fingers. It has long been known that the zinc fingers of basonuclin 1 closely resembled those of the Drosophila disconnected and discorelated proteins, two proteins essential for head development, but the relation between the basonuclins and the disco proteins has remained unclear because the putative function of basonuclin 1 in the control of keratinocyte growth potential appeared unrelated to that of disco and there was no resemblance between basonuclin 1 and Drosophila disco outside of the zinc fingers. The recent generation of a basonuclin-2 knockout has demonstrated that basonuclin 2 shares with disco a function in head development and the availability of new arthropod genome sequences has shown that the basonuclins are the vertebrate orthologs of the insect disco proteins. All these proteins are thought to be transcription factors, and it will have to be determined to what extent they share similar targets.
Asunto(s)
Artrópodos/crecimiento & desarrollo , Artrópodos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Homología de Secuencia de Aminoácido , Vertebrados/crecimiento & desarrollo , Vertebrados/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/genética , Humanos , Datos de Secuencia Molecular , ARN Ribosómico/genética , ARN Ribosómico/metabolismoRESUMEN
Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Adenoviridae/metabolismo , Secuencia de Bases , Inmunoprecipitación de Cromatina , Células Hep G2 , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteonectina/genética , Unión Proteica , Elementos de Respuesta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Regulación hacia Arriba/genéticaRESUMEN
We studied a man with distal hypospadias, partial anomalous pulmonary venous return, mild limb-length inequality and a balanced translocation involving chromosomes 9 and 13. To gain insight into the etiology of his birth defects, we mapped the translocation breakpoints by high-resolution comparative genomic hybridization (CGH), using chromosome 9- and 13-specific tiling arrays to analyze genetic material from a spontaneously aborted fetus with unbalanced segregation of the translocation. The chromosome 13 breakpoint was â¼400 kb away from the nearest gene, but the chromosome 9 breakpoint fell within an intron of Basonuclin 2 (BNC2), a gene that encodes an evolutionarily conserved nuclear zinc-finger protein. The BNC2/Bnc2 gene is abundantly expressed in developing mouse and human periurethral tissues. In all, 6 of 48 unrelated subjects with distal hypospadias had nine novel nonsynonymous substitutions in BNC2, five of which were computationally predicted to be deleterious. In comparison, two of 23 controls with normal penile urethra morphology, each had a novel nonsynonymous substitution in BNC2, one of which was predicted to be deleterious. Bnc2(-/-) mice of both sexes displayed a high frequency of distal urethral defects; heterozygotes showed similar defects with reduced penetrance. The association of BNC2 disruption with distal urethral defects and the gene's expression pattern indicate that it functions in urethral development.