RESUMEN
Mutation of the gene encoding γ-sarcoglycan (SGCG), an integral membrane protein responsible for maintaining the integrity of the muscle cell sarcolemma, results in Limb-Girdle Muscular Dystrophy (LGMD), a congenital disease with no current treatment options. This member of the sarcoglycan glycoprotein family is a vital component of the Dystrophin Complex, which together facilitate normal muscle function. However, very little is known about the structure and dynamics of these proteins, and of membrane glycoproteins in general. This is due to a number of factors, including their complexity, heterogeneity and highly-specific native environments. The expression, purification, and structural study of membrane proteins is further impeded by their hydrophobic nature and consequent propensity to aggregate in aqueous solutions. Here, we report the first successful expression and purification of milligram quantities of full-length recombinant SGCG, utilizing fusion protein-guided overexpression to inclusion bodies in Escherichia coli. Purification of SGCG from the fusion protein, TrpΔLE, was facilitated using chemical cleavage. Cleavage products were then isolated by size-exclusion chromatography. Successful purification of the protein was confirmed using SDS-PAGE and mass spectroscopy. Finally, solution nuclear magnetic resonance spectroscopy of uniformly 15N-labeled SGCG in detergent environments was performed, yielding the first spectra of the full-length membrane glycoprotein, SGCG. These results represent the initial structural studies of SGCG, laying the foundation for further investigation on the interaction and dynamics of other integral membrane proteins. More specifically, this data allows for opportunities in the future for enhanced treatment modalities and cures for LGMD.
Asunto(s)
Sarcoglicanos , Cromatografía en Gel , Clonación Molecular/métodos , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Complejo de Proteínas Asociado a la Distrofina/metabolismo , Escherichia coli , Glicoproteínas/biosíntesis , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Cinturas/etiología , Mutación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sarcoglicanos/biosíntesis , Sarcoglicanos/química , Sarcoglicanos/genética , Sarcoglicanos/aislamiento & purificación , Sarcolema/metabolismo , SolubilidadRESUMEN
Mesoangioblasts have been characterized as a population of vessel-associated stem cells able to differentiate into several mesodermal cell types, including skeletal muscle. Here, we report that the paired box transcription factor Pax3 plays a crucial role in directing mouse mesoangioblasts toward skeletal myogenesis in vitro and in vivo. Mesoangioblasts isolated from the aorta of Pax3 null embryos are severely impaired in skeletal muscle differentiation, whereas most other differentiation programs are not affected by the absence of Pax3. Moreover, Pax3(-/-) null mesoangioblasts failed to rescue the myopathic phenotype of the alpha-sarcoglycan mutant mouse. In contrast, mesoangioblasts from Pax3 gain of function, Pax3(PAX3-FKHR/+), mice display enhanced myogenesis in vitro and are more efficient in regenerating new muscle fibers in this model of muscular dystrophy. These data demonstrate that Pax3 is required for the differentiation of mesoangioblast stem cells into skeletal muscle, in keeping with its role in orchestrating entry into the myogenic program.
Asunto(s)
Vasos Sanguíneos/citología , Diferenciación Celular , Embrión de Mamíferos/citología , Mesodermo/citología , Músculo Esquelético/citología , Factores de Transcripción Paired Box/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Vasos Sanguíneos/enzimología , Huesos/citología , Proliferación Celular , Forma de la Célula , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Ratones , Desarrollo de Músculos , Distrofia Muscular Animal/metabolismo , Miocitos del Músculo Liso/citología , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/deficiencia , Fenotipo , Sarcoglicanos/biosíntesisRESUMEN
Satellite cells refer to resident stem cells in muscle that are activated in response to damage or disease for the regeneration and repair of muscle fibers. The use of stem cell transplantation to treat muscular diseases has been limited by impaired donor cell survival attributed to rejection and an unavailable stem cell niche. We isolated a population of adult muscle mononuclear cells (AMMCs) from normal, strain-matched muscle and transplanted these cells into delta-sarcoglycan-null dystrophic mice. Distinct from other transplant studies, the recipient mice were immunocompetent with an intact endogenous satellite cell pool. We found that AMMCs were 35 times more efficient at restoring sarcoglycan compared with cultured myoblasts. Unlike cultured myoblasts, AMMC-derived muscle fibers expressed sarcoglycan protein throughout their entire length, consistent with enhanced migratory ability. We examined the capacity of single injections of AMMCs to provide long-term benefit for muscular dystrophy and found persistent regeneration after 6 months, consistent with augmentation of the endogenous stem cell pool. Interestingly, AMMCs were more effectively engrafted into aged dystrophic mice for the regeneration of large clusters of sarcoglycan-positive muscle fibers, which were protected from damage, suggesting that the stem cell niche in older muscle remains permissive.
Asunto(s)
Supervivencia de Injerto/fisiología , Leucocitos Mononucleares/trasplante , Músculo Esquelético/citología , Distrofia Muscular Animal/terapia , Trasplante de Células Madre , Animales , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Músculo Esquelético/fisiología , Regeneración , Sarcoglicanos/biosíntesis , Sarcoglicanos/genética , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , TiempoRESUMEN
Duchenne muscular dystrophy (DMD) is a fatal, progressive, muscle-wasting disease caused by defects in the dystrophin. No viral vector except the helper-dependent adenovirus vector (HDAdv) can package 14-kilobase (kb) full-length dystrophin complementary DNA (cDNA), and HDAdv is considerably safer than old-generation adenovirus vectors because of the large-size deletion in its genome. We have generated HDAdv that carries myc-tagged murine full-length dystrophin cDNA (HDAdv-myc-mFLdys). We injected it into multiple proximal muscles of 7-day-old utrophin/dystrophin double knockout mice (dko mice) (which typically show symptoms quite similar to human DMD) because the proximal muscles are affected in DMD patients. Eight weeks after the injections, the transduced dystrophin was widely expressed, and we found a significant reduction in centrally nucleated myofibers and the restoration of the dystrophin-associated proteins, beta-dystroglycan (beta-DG) and alpha-sarcoglycan (alpha-SG), as well as neuronal nitric oxide synthase (nNOS). The injected dko mice also showed an increase in body weight, an improvement in motor performance, and a prolongation of life span. Using HDAdv, we could treat DMD model mice even by transferring the therapeutic gene into multiple skeletal muscles. Our results suggest that multiple intramuscular administrations of HDAdv carrying full-length dystrophin cDNA may reduce symptoms and compensate for lost functions in DMD patients.
Asunto(s)
Distrofina/genética , Distrofina/fisiología , Regulación de la Expresión Génica , Terapia Genética/métodos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Animales , Peso Corporal , Distroglicanos/biosíntesis , Longevidad , Ratones , Ratones Noqueados , Ratones Transgénicos , Destreza Motora , Óxido Nítrico Sintasa de Tipo I/biosíntesis , Sarcoglicanos/biosíntesis , Utrofina/genéticaRESUMEN
The sarcoglycan complex (SGC) is a multimember transmembrane complex interacting with other members of the dystrophin-glycoprotein complex (DGC) to provide a mechanosignaling connection from the cytoskeleton to the extracellular matrix. The SGC consists of four proteins (alpha, beta, gamma, and delta). A fifth sarcoglycan subunit, epsilon-sarcoglycan, shows a wider tissue distribution. Recently, a novel sarcoglycan, the zeta-sarcoglycan, has been identified. All reports about the structure of SGC showed a common assumption of a tetrameric arrangement of sarcoglycans. Addressing this issue, our immunofluorescence and molecular results showed, for the first time, that all sarcoglycans are always detectable in all observed samples. Therefore, one intriguing possibility is the existence of a pentameric or hexameric complex considering zeta-sarcoglycan of SGC, which could present a higher or lower expression of a single sarcoglycan in conformity with muscle type--skeletal, cardiac, or smooth--or also in conformity with the origin of smooth muscle.
Asunto(s)
Músculo Liso/metabolismo , Sarcoglicanos/biosíntesis , Adulto , Técnica del Anticuerpo Fluorescente , Tracto Gastrointestinal/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Complejos Multiproteicos/biosíntesis , Fibras Musculares Esqueléticas/metabolismo , Músculo Liso Vascular/metabolismo , Especificidad de Órganos , Subunidades de Proteína/biosíntesis , Sistema Respiratorio/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema Urogenital/metabolismoRESUMEN
Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.
Asunto(s)
Complejo de Proteínas Asociado a la Distrofina/química , Terapia Genética , Distrofia Muscular de Cinturas/terapia , Oligonucleótidos Antisentido/uso terapéutico , Ingeniería de Proteínas , Sarcoglicanos/genética , Animales , Codón sin Sentido/genética , Diafragma/metabolismo , Diafragma/patología , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Exones , Fibrosis , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Cinturas/genética , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/terapia , Mutación , Miocardio/metabolismo , Miocardio/patología , Oligonucleótidos Antisentido/farmacología , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/química , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/metabolismo , Sarcoglicanos/biosíntesis , Sarcoglicanos/química , Sarcoglicanos/deficiencia , Sarcolema/metabolismo , Eliminación de SecuenciaRESUMEN
Osteonecrosis of the jaw is an adverse outcome associated with bisphosphonate treatment. Bisphosphonates are used in conjunction with antineoplastic chemotherapy for the treatment of hypercalcaemia associated with malignancy, lytic bone metastasis and multiple myeloma. However, it is not known if the osteonecrosis of the jaw lesion originates in the bone or whether it initiates in the gingival epithelium. Two bisphosphonates are commonly used in cancer treatment. One of these is pamidronate disodium, a second-generation bisphosphonate that differs from the first-generation drug because it inhibits bone resorption at a dose that does not affect bone mineralization. The other widely used BP, zoledronate, is a third-generation drug that is the most potent bisphosphonate in clinical use, showing strong anti-osteoclastic activity, similar to pamidronate. The aim of the present study was to evaluate the modifications of human oral mucosa and underlying bone in patients after treatment with these nitrogen-containing bisphosphonates for 24 and 36 months. We analyzed the structural damage of the oral mucosa and damage of the perilesional mandibular bone observing possible correlations from them. Our results allow to express two hypotheses about the mechanism responsible for these results relating to mandible matrix necrosis; first, an increased skeletal microdamage associated with turnover suppression occurred early in treatment and progress with longer treatment duration, second, opening damage in osteonecrosis of the jaw modifies structural morphology of gingival epithelium.
Asunto(s)
Neoplasias Óseas/tratamiento farmacológico , Adhesión Celular/genética , Integrinas/biosíntesis , Osteonecrosis/inducido químicamente , Sarcoglicanos/biosíntesis , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/genética , Calcificación Fisiológica/efectos de los fármacos , Difosfonatos/administración & dosificación , Difosfonatos/efectos adversos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Humanos , Imidazoles/administración & dosificación , Imidazoles/efectos adversos , Inmunohistoquímica , Integrinas/genética , Maxilares/efectos de los fármacos , Maxilares/patología , Maxilares/ultraestructura , Microscopía Electrónica de Rastreo , Mucosa Bucal/efectos de los fármacos , Mucosa Bucal/patología , Mucosa Bucal/ultraestructura , Osteonecrosis/tratamiento farmacológico , Osteonecrosis/patología , Pamidronato , Sarcoglicanos/genética , Ácido ZoledrónicoRESUMEN
Our previous studies have shown that microRNA-383 (miR-383) is one of the most down-regulated miRNA in TGF-ß1-treated mouse ovarian granulosa cells (GC). However, the roles and mechanisms of miR-383 in GC function during follicular development remain unknown. In this study, we found that miR-383 was mainly expressed in GC and oocytes of mouse ovarian follicles. Overexpression of miR-383 enhanced estradiol release from GC through targeting RNA binding motif, single stranded interacting protein 1 (RBMS1). miR-383 inhibited RBMS1 by affecting its mRNA stability, which subsequently suppressed the level of c-Myc (a downstream target of RBMS1). Forced expression of RBMS1 or c-Myc attenuated miR-383-mediated steroidogenesis-promoting effects. Knockdown of the transcription factor steroidogenic factor-1 (SF-1) significantly suppressed the expression of Sarcoglycan zeta (SGCZ) (miR-383 host gene), primary and mature miR-383 in GC, indicating that miR-383 was transcriptionally regulated by SF-1. Luciferase and chromatin immunoprecipitation assays revealed that SF-1 specifically bound to the promoter region of SGCZ and directly transactivated miR-383 in parallel with SGCZ. In addition, SF-1 was involved in regulation of miR-383- and RBMS1/c-Myc-mediated estradiol release from GC. These results suggest that miR-383 functions to promote steroidogenesis by targeting RBMS1, at least in part, through inactivation of c-Myc. SF-1 acts as a positive regulator of miR-383 processing and function in GC. Understanding of regulation of miRNA biogenesis and function in estrogen production will potentiate the usefulness of miRNA in the control of reproduction and treatment of some steroid-related disorders.
Asunto(s)
Estradiol/metabolismo , Células de la Granulosa/metabolismo , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Factor Esteroidogénico 1/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Animales , Femenino , Genes myc , Ratones , Ratones Endogámicos ICR , MicroARNs/biosíntesis , Folículo Ovárico/embriología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Sarcoglicanos/biosíntesis , Sarcoglicanos/genética , Factor Esteroidogénico 1/genética , Transactivadores/antagonistas & inhibidores , Transcripción GenéticaRESUMEN
Colorectal cancers (CRCs) from the suppressor and the mutator carcinogenic pathways display distinctive pathological and clinical features that remain not completely understood. In this context, the aim of this work was to study the differential expression of metalloproteinases and adhesion molecules related to cancer invasiveness in both groups of tumours. We analyzed 84 tissue specimens, 42 primary sporadic CRCs obtained from patients who underwent radical surgery, and its corresponding control tissues. According to microsatellite instability, 31 cancers showed low or null microsatellite instability (MSI-L/MSS) and 11 tumours displayed high microsatellite instability (MSI-H). Expression assays were established using the Oligo GEArray(R) human extracellular matrix and adhesion molecules microarray containing 114 genes. Real-time quantitative PCR (RT-qPCR) confirmed expression data from arrays, using TaqMan probes. Results from oligoarray expression analyses indicated that ITGA3, ITGA9, ITGB4, ITGB7 and MMP15 had significantly higher expression levels in MSI-H tumours versus MSS/MSI-L cancers, whereas COL12A1, CSPG2, FN1, MMP-7 and SGCE were down-regulated in tumours with high microsatellite instability when compared to the stable group. After RT-qPCR validation, two of these genes, MMP-7 and SGCE, were confirmed to have statistical differences between the two groups of tumours studied. In both cases, MSI-H tumours displayed significant lower expression levels than MSI-L/MSS tumours. In conclusion, these two distinctive molecular markers could be related to a diminished invasion in colorectal tumours from the mutator pathway, this may contribute to the understanding of the better patient prognosis conferred by this type of tumours.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 7 de la Matriz/metabolismo , Mutación , Sarcoglicanos/biosíntesis , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Humanos , Inestabilidad de Microsatélites , Repeticiones de Microsatélite/genética , Invasividad Neoplásica , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
BACKGROUND: Alpha-sarcoglycan (alpha-SG) deficiency (limb-girdle muscular dystrophy [LGMD] type 2D) is the most common form of sarcoglycan-LGMD. No treatment is currently available. Prior studies suggest that overexpression of alpha-SG via adeno-associated virus (AAV)-mediated gene transfer results in poorly sustained gene expression related to transgene toxicity. These findings potentially preclude gene therapy as a treatment approach for LGMD2D. METHODS: The human alpha-SG gene (halpha-SG) was directly transferred to the tibialis anterior muscle of 4- to 5-week-old alpha-SG KO mice using AAV, type 1. The gene was placed under control of either the ubiquitously expressed cytomegalovirus (CMV) promoter or muscle specific promoters that included desmin, muscle creatine kinase (MCK), and its further modification, truncated MCK (tMCK). Low (3 x 10(9) vg) and high (3 x 10(10) vg) doses of AAV1.halpha-SG were administered. RESULTS: Sustained gene expression was observed irrespective of promoters at 6 and 12 weeks post gene transfer. Quantitation of alpha-SG gene expression by fiber counts yielded similar levels of myofiber transduction for both MCK promoters (60 to 70%), while 34% of fibers were transduced with the DES promoter. There was a trend toward lower expression at the 12-week time point with the CMV promoter. Western blot analysis revealed alpha-SG overexpression using CMV and both the MCK promoters. CONCLUSION: Our data demonstrate robust and sustained adeno-associated virus type 1 alpha-sarcoglycan gene expression under control of muscle creatine kinase promoters, without evidence of cytotoxicity. These findings support the use of gene therapy as a potential treatment approach for limb-girdle muscular dystrophy type 2D.
Asunto(s)
Técnicas de Transferencia de Gen/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/genética , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/terapia , Sarcoglicanos/genética , Animales , Ensayos Clínicos como Asunto/normas , Creatina Quinasa/genética , Citomegalovirus/genética , Dependovirus/genética , Expresión Génica/genética , Terapia Genética/efectos adversos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Distrofia Muscular de Cinturas/fisiopatología , Miositis/genética , Miositis/inmunología , Miositis/metabolismo , Regiones Promotoras Genéticas/genética , Sarcoglicanos/biosíntesis , Sarcoglicanos/deficiencia , Resultado del Tratamiento , Regulación hacia Arriba/genéticaRESUMEN
Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy (FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated alpha-dystroglycan, together with reduced expression of beta-dystroglycan and alpha-, beta-, gamma-, delta-sarcoglycans. Although the expression of glycosylated alpha-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of alpha-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P>0.1 by the two-tailed t test). This study implied either O-linked glycosylation defects of alpha-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated alpha-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces.