RESUMO
Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. GORAB, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the GorabNull full knockout, Gorab was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only GorabPrx1 and GorabRunx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of GorabNull mutants and in bone of GorabPrx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from GorabNull mutants. In bone from GorabPrx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured GORAB-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-ß in GorabPrx1 bone tissue leading to enhanced downstream signalling, which was reproduced in GORAB-deficient fibroblasts. Our data suggest that the loss of Gorab primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.
Assuntos
Doenças Ósseas/congênito , Nanismo/metabolismo , Osteoblastos/patologia , Proteoglicanas/metabolismo , Dermatopatias Genéticas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Diferenciação Celular , Decorina/metabolismo , Dermatan Sulfato/metabolismo , Modelos Animais de Doenças , Nanismo/patologia , Feminino , Fraturas Ósseas/genética , Glicosilação , Proteínas da Matriz do Complexo de Golgi , Células-Tronco Mesenquimais/patologia , Células-Tronco Mesenquimais/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/metabolismo , Transdução de Sinais , Dermatopatias Genéticas/patologia , Proteínas de Transporte Vesicular/genéticaRESUMO
Osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS) are variable genetic disorders that overlap in different ways [Cole 1993; Grahame 1999]. Here, we describe a boy presenting with severe muscular hypotonia, multiple fractures, and joint hyperflexibility, features that are compatible with mild OI and hypermobility type EDS, respectively. By whole exome sequencing, we identified both a COL1A1 mutation (c.4006-1G > A) inherited from the patient's mildly affected mother and biallelic missense variants in TNXB (p.Val1213Ile, p.Gly2592Ser). Analysis of cDNA showed that the COL1A1 splice site mutation led to intron retention causing a frameshift (p.Phe1336Valfs*72). Type 1 collagen secretion by the patient's skin fibroblasts was reduced. Immunostaining of a muscle biopsy obtained from the patient revealed a clear reduction of tenascin-X in the extracellular matrix compared to a healthy control. These findings imply that the combination of the COL1A1 mutation with the TNXB variants might cause the patient's unique phenotype.
Assuntos
Colágeno Tipo I/genética , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/genética , Heterozigoto , Mutação , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/genética , Fenótipo , Tenascina/genética , Alelos , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Análise Mutacional de DNA , Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Lactente , Masculino , LinhagemRESUMO
Autosomal recessive cutis laxa (ARCL) syndromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debré type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2 subunit of the V-type H(+)-ATPase, playing a role in proton translocation, and possibly also in membrane fusion. Here, we describe a highly variable phenotype in 13 patients with ARCL2, including the oldest affected individual described so far, who showed strikingly progressive dysmorphic features and heterotopic calcifications. In these individuals we identified 17 ATP6V0A2 mutations, 14 of which are novel. Furthermore, we demonstrate a localization of ATP6V0A2 at the Golgi-apparatus and a loss of the mutated ATP6V0A2 protein in patients' dermal fibroblasts. Investigation of brefeldin A-induced Golgi collapse in dermal fibroblasts as well as in HeLa cells deficient for ATP6V0A2 revealed a delay, which was absent in cells deficient for the ARCL-associated proteins GORAB or PYCR1. Furthermore, fibroblasts from patients with ATP6V0A2 mutations displayed elevated TGF-ß signalling and increased TGF-ß1 levels in the supernatant. Our current findings expand the genetic and phenotypic spectrum and suggest that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ATP6V0A2-related ARCL.
Assuntos
Cútis Laxa/congênito , Mutação/genética , ATPases Translocadoras de Prótons/genética , Fator de Crescimento Transformador beta1/metabolismo , Adolescente , Adulto , Apoptose , Western Blotting , Brefeldina A/farmacologia , Células Cultivadas , Pré-Escolar , Cútis Laxa/genética , Cútis Laxa/metabolismo , Cútis Laxa/patologia , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Imunofluorescência , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Lactente , Masculino , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Adulto JovemRESUMO
Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125-148; Singla, V., and J.F. Reiter. 2006. Science. 313:629-633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517-524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.
Assuntos
Cílios/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Epitélio Pigmentado Ocular/citologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
One of the characteristics of the mammalian Golgi is its position adjacent to the nucleus. This characteristic is maintained through the action of the microtubule (MT) minus end-directed motor dynein and MT-associated proteins (MAPs). Recent findings suggest that GMAP-210, a member of the golgin family of proteins, may help to link Golgi membranes and vesicles with the MT cytoskeleton. However, there are good grounds to doubt that either GMAP-210 or its yeast homologue Rud3p is a MAP. Instead, they appear to function in vesicle trafficking events at the Golgi together with the GTPase ARF1 and a small membrane protein, Erv14. As such, the interesting question of how the Golgi interacts with MTs may well remain open to further investigation.
Assuntos
Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Vesículas Transportadoras/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Proteínas do Citoesqueleto , Humanos , Proteínas de Membrana/metabolismo , Proteínas Nucleares , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Gerodermia osteodysplastica is a hereditary segmental progeroid disorder affecting skin, connective tissues, and bone that is caused by loss-of-function mutations in GORAB. The golgin, RAB6-interacting (GORAB) protein localizes to the Golgi apparatus and interacts with the small GTPase RAB6. In this study, we used different approaches to shed more light on the recruitment of GORAB to this compartment. We show that GORAB best colocalizes with trans-Golgi markers and is rapidly displaced upon Brefeldin A exposition, indicating a loose association with Golgi membranes. A yeast two-hybrid screening revealed a specific interaction with the small GTPase ADP-ribosylation factor (ARF5) in its active, GTP-bound form. ARF5 and RAB6 bind to GORAB via the same internal Golgi-targeting RAB6 and ARF5 binding (IGRAB) domain. Two GORAB missense mutations identified in gerodermia osteodysplastica patients fall within this IGRAB domain. GORAB carrying the mutation p.Ala220Pro had a cytoplasmic distribution and failed to interact with both RAB6 and ARF5. In contrast, the p.Ser175Phe mutation displaced GORAB from the Golgi compartment to vesicular structures and selectively impaired ARF5 binding. Our findings indicate that the IGRAB domain is crucial for the Golgi localization of GORAB and that loss of this localization impairs its physiological function.
Assuntos
Fatores de Ribosilação do ADP/genética , Mutação de Sentido Incorreto , Ligação Proteica/genética , Proteínas rab de Ligação ao GTP/genética , Doenças Ósseas/congênito , Doenças Ósseas/genética , Doenças Ósseas/fisiopatologia , Células Cultivadas , Nanismo/genética , Nanismo/fisiopatologia , Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa/metabolismo , Humanos , Sensibilidade e Especificidade , Dermatopatias Genéticas/genética , Dermatopatias Genéticas/fisiopatologia , TransfecçãoRESUMO
Gerodermia osteodysplastica is an autosomal recessive disorder characterized by wrinkly skin and osteoporosis. Here we demonstrate that gerodermia osteodysplastica is caused by loss-of-function mutations in SCYL1BP1, which is highly expressed in skin and osteoblasts. The protein localizes to the Golgi apparatus and interacts with Rab6, identifying SCYL1BP1 as a golgin. These results associate abnormalities of the secretory pathway with age-related changes in connective tissues.