RESUMEN
Background: Huntington's disease is an inheritable autosomal dominant disorder caused by an expanded CAG trinucleotide repeat within the Huntingtin gene, leading to a polyglutamine (polyQ) expansion in the mutant protein. Objective: A potential therapeutic approach for delaying or preventing the onset of the disease involves enhancing the degradation of the aggregation-prone polyQ-expanded N-terminal mutant huntingtin (mHTT) exon1 fragment. A few proteases and peptidases have been identified that are able to cleave polyQ fragments with low efficiency. This study aims to identify a potent polyQ-degrading endopeptidase. Methods: Here we used quenched polyQ peptides to identify a polyQ-degrading endopeptidase. Next we investigated its role on HTT turnover, using purified polyQ-expanded HTT fragments and striatal cells expressing mHTT exon1 peptides. Results: We identified insulin-degrading enzyme (IDE) as a novel endopeptidase for degrading polyQ peptides. IDE was, however, ineffective in reducing purified polyQ-expanded HTT fragments. Similarly, in striatal cells expressing mHTT exon1 peptides, IDE did not enhance mHTT turnover. Conclusions: This study shows that despite IDE's efficiency in degrading polyQ peptides, it does not contribute to the direct degradation of polyQ-expanded mHTT fragments.
Asunto(s)
Proteína Huntingtina , Insulisina , Péptidos , Insulisina/metabolismo , Insulisina/genética , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Péptidos/metabolismo , Humanos , Animales , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Cuerpo Estriado/metabolismoRESUMEN
The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of many cytoplasmic and nuclear proteins. The proteasome is essential and proteasome malfunction is associated with various disease pathologies. Proteasome activity depends on its catalytic subunits which are interchangeable and also on the interaction with the associated regulatory cap complexes. Here, we describe and compare various methods that allow the study of proteasome function in living cells. Methods include the use of fluorescently tagged proteasome subunits and the use of activity-based proteasome probes. These probes can be used in both biochemical assays and in microscopy-based experiments. Together with tagged proteasomes, they can be used to study proteasome localization, dynamics, and activity.
RESUMEN
MPP3 and CRB1 both interact directly with PALS1/MPP5 and through this scaffold protein may form a large protein complex. To investigate the role of MPP3 in the retina we have analyzed conditional mutant Mpp3 knockout mice. Ultrastructural localization studies revealed that MPP3 is predominantly localized in apical villi of Müller glia cells. Retinas lacking MPP3 developed late onset retinal degeneration, with sporadic foci of rosette formation in the central part of the retina. Retinal degeneration in Mpp3 cKO mice was accelerated by exposure to moderate levels of white light. Electroretinography recordings in aging mice under both scotopic and photopic conditions ranged from normal to mildly subnormal, while the magnitude correlated with the strength and extent of morphological alterations. Loss of MPP3 resulted in significant loss of PALS1 at the subapical region adjacent to adherens junctions, and loss of MPP3 in Pals1 conditional knockdown retinas significantly accelerated the onset of retinal degeneration. These data suggest that MPP3 is required for maintaining proper levels of PALS1 at the subapical region, and indicate that the MPP3 gene is a candidate modulator of the Crumbs complex.
Asunto(s)
Adhesión Celular/fisiología , Células Ependimogliales/metabolismo , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Células Fotorreceptoras/metabolismo , Animales , Cateninas/metabolismo , Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Electrorretinografía , Células Ependimogliales/ultraestructura , Angiografía con Fluoresceína , Regulación de la Expresión Génica/genética , Guanilato-Quinasas/deficiencia , Luz/efectos adversos , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Inmunoelectrónica , Nectinas , Células Fotorreceptoras/ultraestructura , Retina/citología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Tomografía de Coherencia Óptica , Vías Visuales/metabolismo , Catenina deltaRESUMEN
During mammalian cortical development, division of progenitor cells occurs at the apical ventricular zone. Apical complex proteins and adherens junctions regulate the different modes of division. Here, we have identified the membrane-associated guanylate kinase protein membrane palmitoylated protein 3 (MPP3) as an essential protein for the maintenance of these complexes. MPP3 localizes at the apical membrane in which it shows partial colocalization with adherens junction proteins and apical proteins. We generated Mpp3 conditional knock-out mice and specifically ablated Mpp3 expression in cortical progenitor cells. Conditional deletion of Mpp3 during cortical development resulted in a gradual loss of the apical complex proteins and disrupted adherens junctions. Although there is cellular disorganization in the ventricular zone, gross morphology of the cortex was unaffected during loss of MPP3. However, in the ventricular zone, removal of MPP3 resulted in randomization of spindle orientation and ectopically localized mitotic cells. Loss of MPP3 in the developing cortex resulted in delayed migration of progenitor cells, whereas the rate of cell division and exit from the cell cycle was not affected. This resulted in defects in cortical stratification and ectopically localized layer II-IV pyramidal neurons and interneurons. These data show that MPP3 is required for maintenance of the apical protein complex and adherens junctions and for stratification and proper migration of neurons during the development of the cortex.
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Movimiento Celular/genética , Corteza Cerebral , Regulación del Desarrollo de la Expresión Génica/genética , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Proliferación Celular , Corteza Cerebral/citología , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Ventrículos Cerebrales/citología , Ventrículos Cerebrales/embriología , Ventrículos Cerebrales/crecimiento & desarrollo , Embrión de Mamíferos , Guanilato-Quinasas/deficiencia , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurogénesis/genéticaRESUMEN
In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells.
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Proteínas del Ojo/genética , Proteínas de la Membrana/genética , Mutación , Proteínas del Tejido Nervioso/genética , Retina/metabolismo , Retinitis Pigmentosa/genética , Animales , Apoptosis , Secuencia de Bases , Cartilla de ADN , Electrorretinografía , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Fotorreceptoras de Vertebrados/patología , Reacción en Cadena de la Polimerasa , Retina/crecimiento & desarrollo , Retinitis Pigmentosa/patología , Tomografía de Coherencia ÓpticaRESUMEN
Most cytoplasmic and nuclear proteins are degraded via the ubiquitin-proteasome system into peptides, which are subsequently hydrolyzed by downstream aminopeptidases. Inefficient degradation can lead to accumulation of protein fragments, and subsequent aggregation and toxicity. Whereas the role of the proteasome and the effect of its impairment on aggregation have been intensively studied, little is known about how cells deal with peptides that show resistance to degradation by aminopeptidases. Here, we introduced peptidase-resistant peptides into living cells and show that these peptides rapidly and irreversibly accumulate into puncta in the perinuclear region of the cell. Accumulation appears to be independent of peptide sequence but is less efficient for longer peptides. The puncta colocalize with autophagosomal and lysosomal markers, suggesting that these peptides end up within lysosomes via macroautophagy. Surprisingly, the peptides still accumulate within lysosomes when macroautophagy is impaired, suggesting a trafficking route independent of macroautophagy. Upon lysosomal uptake, peptides are degraded, suggesting that cells can clear peptidase-resistant proteasomal products by an alternative pathway, which targets them to lysosomes.
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Aminopeptidasas/metabolismo , Lisosomas/metabolismo , Péptidos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Citoplasma/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Humanos , Melanoma/metabolismo , Fagosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Isoformas de Proteínas , ProteolisisRESUMEN
Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal blinding diseases, such as Leber congenital amaurosis and retinitis pigmentosa. In the previous studies we have shown that Crb1 resides in retinal Müller glia cells and that loss of Crb1 results in retinal degeneration (particularly in the inferior temporal quadrant of the mouse eye). Degeneration is increased by exposure to white light. Here, we studied the role of light and aging to gain a better understanding of the factors involved in the progress of retinal disease. Our data reveal that light is neither sufficient nor required to induce retinal disorganization and degeneration in young Crb1(-/-) mutant mice, suggesting that it rather modulates the retinal phenotype. Gene expression profiling showed that expression of five genes is altered in light-exposed Crb1(-/-) mutant retinas. Three of the five genes are involved in chromosome stabilization (Pituitary tumor transforming gene 1 or Pttg1, Establishment of cohesion 1 homolog 1 or Esco1, and a gene similar to histone H2B). In aged retinas, degeneration of photoreceptors, inner retinal neurons, and retinal pigment epithelium was practically limited to the inferior temporal quadrant. Loss of Crb1 in Müller glia cells resulted in an irregular number and size of their apical villi. We propose that Crb1 is required to regulate number and size of these Müller glia cell villi. The subsequent loss of retinal integrity resulted in neovascularization, in which blood vessels of the choroid protruded into the neural retina.