Distroglicanopatías: estudios genéticos y funcionales de la glicosiltransferasa POMGNT1. Caracterización glicoproteómica del alfa-distroglicano nativo de ratón

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 27 de Junio de 201

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

Skp1‐Cul1‐F‐box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma‐associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F‐box and leucine‐rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17‐mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17–Sufu axis in the pathogenesis of medulloblastoma

A double homozygous mutation in the POMT1 gene involving exon skipping gives rise to Walker-Warburg syndrome in two Spanish gypsy families

Carta al editor.-- El pdf es la versión post-print sin figuras.-- et al.This work was supported by the Spanish Fondo de Investigaciones Sanitarias’ grant PI06/0378.Peer Reviewe

O-mannose and O-N-acetyl galactosamine glycosylation of mammalian α-dystroglycan is conserved in a region-specific manner

Defects in the O-linked glycosylation of the peripheral membrane protein α-dystroglycan (α-DG) are the main cause of several forms of congenital muscular dystrophies and thus the characterization of the glycosylation of α-DG is of great medical importance. A detailed investigation of the glycosylation pattern of the native α-DG protein is essential for the understanding of the biological processes related to human disease in which the protein is involved. To date, several studies have reported novel O-glycans and attachment sites on the mucin-like domain of mammalian α-DG with both similar and contradicting glycosylation patterns, indicating the species-specific O-glycosylation of mammalian α-DG. By applying a standardized purification scheme and subsequent glycoproteomic analysis of native α-DG from rabbit and human skeletal muscle biopsies and from cultured mouse C2C12 myotubes, we show that the O-glycosylation patterns of the mucin-like domain of native α-DG are conserved among mammalians in a region-specific manner. © 2012 The Author.This work was supported by funds from grants from Laboratory Medicine, Sahlgrenska University Hospital, and Governmental grants to the Sahlgrenska University Hospital and by grants from the Swedish Research Council (No.8266), the IngaBritt and Arne Lundberg Foundation and from the Fondo de Investigaciones Sanitarias (PI09/0343).Peer Reviewe

Clinical features and molecular characterization of a patient with muscle-eye-brain disease: A novel mutation in the POMGNT1 gene

Muscle-eye-brain disease is a congenital muscular dystrophy characterized by structural brain and eye defects. Here, we describe a 12-year-old boy with partial agenesis of corpus callosum, ventriculomegaly, flattened brain stem, diffuse pachygyria, blindness, profound cognitive deficiencies, and generalized muscle weakness, yet without a clear dystrophic pattern on muscle biopsy. There was no glycosylation of α-dystroglycan and the genetic screening revealed a novel truncating mutation, c.1545delC (p.Tyr516Thrfs*21), and a previously identified missense mutation, c.1469G>A (p.Cys490Tyr), in the protein O-mannose beta-1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) gene. These findings broaden the clinical spectrum of muscle-eye-brain disease to include pronounced hypotonia with severe brain and eye malformations, yet with mild histopathologic changes in the muscle specimen, despite the absence of glycosylated α-dystroglycan.Peer Reviewe

Inmunolocalización en la retina humana de POMT1 y POMT2, dos O-manosil transferasas de proteínas asociadas al síndrome de Walker-Warburg

Resumen del póster presentado al XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre de 2011.Las distroglicanopatías son un grupo de distrofias musculares recesivas raras que afectan con mayor o menor gravedad al músculo, ojo y cerebro. Se caracterizan por una hipoglicosilación del α-distroglicano (α-DG), una proteína implicada en el anclaje celular a la lámina basal y el establecimiento de sinapsis en el SNC. La más severa es el síndrome de Walker-Warburg, que conlleva graves defectos cerebrales y oculares y está causado principalmente por mutaciones en el gen POMT1, y con menor frecuencia en POMT2. Estos genes codifican las proteína O-manosil transferasas-1 y -2 responsables de glicosilar el α-DG. Hasta la fecha no se ha estudiado su expresión en la retina de mamíferos adultos. En este trabajo describimos que el gen POMT1 se expresa a nivel de mRNA y proteína en la retina adulta de todos los mamíferos estudiados, desde roedores hasta la especie humana. Hemos detectado asimismo diversas isoformas putativas de POMT1. También hemos investigado el patrón de inmunolocalización de POMT1 y POMT2 en los distintos tipos neuronales y gliales de la retina humana. Hemos determinado que ambas proteínas se colocalizan en las células gliales de Müller. POMT1 y POMT2 también se observan en fotorreceptores (segmentos externos e internos), así como en algunas células bipolares, amacrinas y ganglionares. Dado el amplio patrón de expresión de POMT1 y POMT2, proponemos que estas proteínas podrían desempeñar otras funciones en la retina adulta humana adicionales a su papel descrito en la Oglicosilación del α-DG.Instituto de Salud Carlos III PI09/1623 (J.M.N.) y PI09/0343 (J.C.).Peer Reviewe

O-mannose and O-N-acetyl galactosamine glycosylation of mammalian α-dystroglycan is conserved in a region-specific manner

Defects in the O-linked glycosylation of the peripheral membrane protein α-dystroglycan (α-DG) are the main cause of several forms of congenital muscular dystrophies and thus the characterization of the glycosylation of α-DG is of great medical importance. A detailed investigation of the glycosylation pattern of the native α-DG protein is essential for the understanding of the biological processes related to human disease in which the protein is involved. To date, several studies have reported novel O-glycans and attachment sites on the mucin-like domain of mammalian α-DG with both similar and contradicting glycosylation patterns, indicating the species-specific O-glycosylation of mammalian α-DG. By applying a standardized purification scheme and subsequent glycoproteomic analysis of native α-DG from rabbit and human skeletal muscle biopsies and from cultured mouse C2C12 myotubes, we show that the O-glycosylation patterns of the mucin-like domain of native α-DG are conserved among mammalians in a region-specific manner. © 2012 The Author.This work was supported by funds from grants from Laboratory Medicine, Sahlgrenska University Hospital, and Governmental grants to the Sahlgrenska University Hospital and by grants from the Swedish Research Council (No.8266), the IngaBritt and Arne Lundberg Foundation and from the Fondo de Investigaciones Sanitarias (PI09/0343).Peer Reviewe

A duplicated sequence in the POMGNT1 gene promoter causes its transcriptional repression in limb-girdle muscular dystrophy type 2O

Póster presentado al XXXI Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre de 2011.-- et al.Limb-girdle muscular dystrophy type 2O (LGMD2O) belongs to a group of rare muscular dystrophies named dystroglycanopathies, which are molecularly characterized by hypoglycosylation of α-dystroglycan and present a wide clinical spectrum ranging from congenital muscular dystrophy associated with severe brain and eye malformation to mild forms of muscle weakness with later onset and without brain involvement. We report the first mutation in the promoter region of POMGNT1 gene in a LGMD2O patient, consisting in a 9-bp duplication in homozygous state. By analyzing the downstream effects of this alteration we found a decreased expression of POMGNT1 mRNA and protein in our patient due to a negative regulation exerted by ZFN202 transcription factor. By functional analysis of a series of deletion constructs we localized the proximal promoter region between -402 and +644 bp relative to the transcription start site and found a 75% decrease of luciferase activity of the construct carrying the new mutation compared with the wild type construct. Electrophoretic mobility shift assay revealed that this region contains three Sp1, one Ets1 and one GATA transcription factor binding sites. The three Sp1 sites were confirmed by chromatin immunoprecipitation assay. Surprisingly, the reported mutation generated an additional ZNF202 binding site and caused a strong binding of this transcriptional repressor to the mutated POMGNT1 promoter, whereas it did not recognize the wild type promoter. These studies underline the importance of the promoter regions for adequate gene expression and the association of certain types of dystroglycanopathies with the impairment of yet-unknown regulatory mechanisms.Funding: Instituto de Salud Carlos III PI09/0343 (J.C) and PI09/1623 (J.M.N).Peer Reviewe

Generación de knock-outs de FKTN y FKRP en la línea muscular C2C12 de ratón mediante la tecnología “TALEN” para el estudio de distroglicanopatías

Póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Mutaciones en los genes FKTN (fukutin) y FKRP (fukutin-related protein), que codifican dos proteínas muy relacionadas, son causantes de un conjunto de enfermedades congénitas de carácter recesivo denominadas actualmente como distrofias musculares-distroglicanopatías (MDDG, de sus siglas en inglés). La falta de función de estas proteínas conduce a la hipoglicosilación del alfa-distroglicano (α-DG). Esta proteína sirve de anclaje de la célula a la matriz extracelular (MEC) en diferentes órganos y tejidos, sobre todo músculo, cerebro y retina. El α-DG sufre una importante y masiva O-glicosilación postraduccional en residuos de serina/treonina de su conocido “dominio mucina”, generando diferentes tipos de cadenas tanto de O-manosilglicanos como de O-N-acetil-galactosaminilglicanos. A través de estos residuos se realiza la interacción con proteínas de la MEC como laminina, agrina y perlecano, así como con las proteínas sinápticas neurexina y pikachurina. Sin embargo, hasta la fecha, la función de estas proteínas FKTN y FKRP en la O-glicosilación del α-DG sigue siendo desconocida. En un intento de elucidar sus funciones, hemos generado una serie de mutantes knock-out (KO), tanto para FKTN como para FKRP en la línea celular C2C12 de mioblastos de ratón, utilizando la tecnología “TALEN” (transcription activator-like effector endonuclease). Un número significativo de estos mutantes KO son defectivos en la O-glicosilación del α-DG, tras ser analizados mediante western blotting y citometría de flujo, utilizando anticuerpos contra las cadenas glicosílicas del α-DG. El objetivo final de este estudio es analizar mediante glicómica comparada los residuos glicosílicos afectados en el α-DG por la falta de función de ambas proteínas frente a la línea celular C2C12 silvestre.Instituto de Salud Carlos III, proyecto PI12/0157.Peer reviewe