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1.
Neuromuscul Disord ; 28(2): 158-168, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29305133

RESUMO

GNE myopathy is a rare distal myopathy, caused by mutations in the GNE gene, affecting sialic acid synthesis. Clinical presentation varies from asymptomatic early stage patients to severely debilitating forms. This first report describes clinical presentations and severity of the disease, using data of 150 patients collected via the on-line, patient-reported registry component of the GNE Myopathy Disease Monitoring Program (GNEM-DMP). Disease progression was prospectively analysed, over a 2-year period, using the GNE myopathy functional activity scale (GNEM-FAS). The average annual rates of decline in function were estimated at -9.6% and -3.2% in ambulant and non-ambulant patients respectively. 4.3% of participants became non-ambulant within one year. The mean time from onset to required use of a wheelchair was 11.9 years. Mean delay of genetic diagnosis from symptom onset was 5.2 years. Mutation specific analysis demonstrated genotype-phenotype relationships; i.e. p.Ala662Val may be associated with a more severe phenotype, compared to p.Val727Met. Patients with compound heterozygous mutation in epimerase and kinase domain appeared to have a more severe phenotype compared to patients with both mutations located within one domain. Acknowledging the limitations of the study, these findings suggest that the severity of the GNE mutations affects disease severity. The GNEM-DMP is a useful data collection tool, prospectively measuring the progression of GNE myopathy, which could play an important role in translational and clinical research and further understanding of genotype-phenotype correlations.

2.
PLoS One ; 12(3): e0173261, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28267778

RESUMO

GNE myopathy (GNEM), also known as hereditary inclusion body myopathy (HIBM), is a late- onset, progressive myopathy caused by mutations in the GNE gene encoding the enzyme responsible for the first regulated step in the biosynthesis of sialic acid (SA). The disease is characterized by distal muscle weakness in both the lower and upper extremities, with the quadriceps muscle relatively spared until the late stages of disease. To explore the role of SA synthesis in the disease, we conducted a comprehensive and systematic analysis of both free and total SA levels in a large cohort of GNEM patients and a mouse model. A sensitive LC/MS/MS assay was developed to quantify SA in serum and muscle homogenates. Mean serum free SA level was 0.166 µg/mL in patients and 18% lower (p<0.001) than that of age-matched control samples (0.203 µg/mL). In biopsies obtained from patients, mean free SA levels of different muscles ranged from 0.046-0.075 µg/µmol Cr and were markedly lower by 72-85% (p<0.001) than free SA from normal controls. Free SA was shown to constitute a small fraction (3-7%) of the total SA pool in muscle tissue. Differences in mean total SA levels in muscle from patients compared with normal controls were less distinct and more variable between different muscles, suggesting a small subset of sialylation targets could be responsible for the pathogenesis of GNEM. Normal quadriceps had significantly lower levels of free SA (reduced by 39%) and total SA (reduced by 53%) compared to normal gastrocnemius. A lower SA requirement for quadriceps may be linked to the reported quadriceps sparing in GNEM. Analysis of SA levels in GneM743T/M743T mutant mice corroborated the human study results. These results show that serum and muscle free SA is severely reduced in GNEM, which is consistent with the biochemical defect in SA synthesis associated with GNE mutations. These results therefore support the approach of reversing SA depletion as a potential treatment for GNEM patients.


Assuntos
Miopatias Distais/metabolismo , Músculo Esquelético/metabolismo , Ácido N-Acetilneuramínico/deficiência , Adolescente , Adulto , Idoso , Animais , Biomarcadores , Biópsia , Cromatografia Líquida , Modelos Animais de Doenças , Miopatias Distais/sangue , Miopatias Distais/patologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Ácido N-Acetilneuramínico/sangue , Espectrometria de Massas em Tandem , Adulto Jovem
3.
Mov Disord ; 31(11): 1694-1703, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27535350

RESUMO

BACKGROUND: Myoclonus-dystonia is a neurogenic movement disorder caused by mutations in the gene encoding ɛ-sarcoglycan. By contrast, mutations in the α-, ß-, γ-, and δ-sarcoglycan genes cause limb girdle muscular dystrophies. The sarcoglycans are part of the dystrophin-associated protein complex in muscle that is disrupted in several types of muscular dystrophy. Intriguingly, patients with myoclonus-dystonia have no muscle pathology; conversely, limb-girdle muscular dystrophy patients have not been reported to have dystonia-associated features. To gain further insight into the molecular mechanisms underlying these differences, we searched for evidence of a sarcoglycan complex in the brain. METHODS: Immunoaffinity chromatography and mass spectrometry were used to purify ubiquitous and brain-specific ɛ-sarcoglycan directly from tissue. Cell models were used to determine the effect of mutations on the trafficking and assembly of the brain sarcoglycan complex. RESULTS: Ubiquitous and brain-specific ɛ-sarcoglycan isoforms copurify with ß-, δ-, and ζ-sarcoglycan, ß-dystroglycan, and dystrophin Dp71 from brain. Incorporation of a muscular dystrophy-associated ß-sarcoglycan mutant into the brain sarcoglycan complex impairs the formation of the ßδ-sarcoglycan core but fails to abrogate the association and membrane trafficking of ɛ- and ζ-sarcoglycan. CONCLUSIONS: ɛ-Sarcoglycan is part of the dystrophin-associated protein complex in brain. Partial preservation of ɛ- and ζ-sarcoglycan in brain may explain the absence of myoclonus dystonia-like features in muscular dystrophy patients. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.


Assuntos
Encéfalo/metabolismo , Distúrbios Distônicos/metabolismo , Distrofias Musculares/metabolismo , Sarcoglicanas/metabolismo , Animais , Células HEK293 , Humanos , Ratos
4.
Hum Genet ; 132(8): 923-34, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23591631

RESUMO

Dystroglycanopathies are characterized by a reduction in the glycosylation of alpha-dystroglycan (α-DG). A common cause for this subset of muscular dystrophies is mutations in the gene of fukutin-related protein (FKRP). FKRP mutations have been associated with a wide spectrum of clinical severity from severe Walker-Warburg syndrome and muscle-eye-brain disease with brain and eye defects to mild limb-girdle muscular dystrophy 2I with myopathy only. To examine the affects of FKRP mutations on the severity of the disease, we have generated homozygous and compound heterozygous mouse models with human mutations in the murine FKRP gene. P448Lneo+ and E310delneo+ mutations result in severe dystrophic and embryonic lethal phenotypes, respectively. P448Lneo+/E310delneo+ compound heterozygotes exhibit brain defects and severe muscular dystrophies with near absence of α-DG glycosylation. Removal of the Neo(r) cassette from the P448Lneo+ homozygous mice eliminates overt brain and eye defects, and reduces severity of dystrophic phenotypes. Furthermore, introduction of the common L276I mutation to generate transgenic L276Ineo+ homozygous and L276Ineo+/P448Lneo+ and L276Ineo+/E310delneo+ compound heterozygotes results in mice displaying milder dystrophies with reduced α-DG glycosylation and no apparent brain defects. Limited sampling and variation in functionally glycosylated α-DG levels between and within muscles may explain the difficulties in correlating FKRP expression levels with phenotype in clinics. The nature of individual mutations, expression levels and status of muscle differentiation all contribute to the phenotypic manifestation. These mutant FKRP mice are useful models for the study of disease mechanism(s) and experimental therapies.


Assuntos
Modelos Animais de Doenças , Distrofias Musculares/classificação , Distrofias Musculares/patologia , Proteínas/fisiologia , Animais , Western Blotting , Feminino , Imunofluorescência , Glicosilação , Heterozigoto , Homozigoto , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distrofias Musculares/etiologia , Mutação , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
J Biol Chem ; 287(12): 9560-7, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22270369

RESUMO

α-Dystroglycan (α-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of α-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-α2,3-Gal-ß1,4-GlcNAc-ß1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of α-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, FKRP (fukutin-related protein) is also involved in the post-phosphoryl modification of O-mannose on α-DG. Furthermore, we have found that the glycosylation status of α-DG in lung and testis is minimally affected by defects in fukutin, LARGE, or FKRP. α-DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α-DG functional expression as a laminin receptor in normal tissues and cells.


Assuntos
Distroglicanas/metabolismo , Laminina/metabolismo , Distrofias Musculares/metabolismo , Animais , Modelos Animais de Doenças , Distroglicanas/genética , Feminino , Humanos , Laminina/genética , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Distrofias Musculares/genética , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/genética , Proteínas/metabolismo , Testículo/metabolismo
6.
Brain Pathol ; 21(6): 699-704, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21507121

RESUMO

The second International Workshop for Glycosylation Defects in Muscular Dystrophies took place on November 11 and 12, 2010 in Charlotte, North Carolina, USA. The meeting was hosted by the Carolinas Medical Center with financial support from the Carolinas Muscular Dystrophy Research Endowment at the Carolinas HealthCare Foundation, the Muscular Dystrophy Association and funds raised by the "Jeans, Genes & Geniuses" event organized by Jane and Luther Lockwood. Since conducting the first workshop in May 2008, significant progress has been made in a subset of muscular dystrophies associated with defects in alpha-dystroglycan (α-DG) glycosylation. New findings on α-DG glycosylation and creation of novel animal models have expanded our understanding of the disease mechanism. The 2010 workshop focused on the following topics; (i) functional glycosylation of α-DG; (ii) animal models; and (iii) novel experimental therapies. The workshop brought together a total of 22 internationally renowned scientists and clinicians from US, UK, Denmark and Japan with active research and expertise in these areas. Overall, the workshop provided a unique opportunity to discuss the significance of recent progress, facilitate international collaboration, and identify new approaches to treat the disease.


Assuntos
Distrofias Musculares/metabolismo , Animais , Glicosilação , Humanos , Distrofias Musculares/fisiopatologia , Distrofias Musculares/terapia
7.
Am J Pathol ; 178(1): 261-72, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21224063

RESUMO

Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology.


Assuntos
Técnicas de Silenciamento de Genes/métodos , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Proteínas/genética , Interferência de RNA , Adenoviridae , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Distroglicanas/metabolismo , Vetores Genéticos , Glicosilação , Camundongos , Camundongos Endogâmicos ICR , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , RNA Interferente Pequeno/genética
8.
Hum Mol Genet ; 19(20): 3995-4006, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20675713

RESUMO

Mutations in fukutin-related protein (FKRP) cause a common subset of muscular dystrophies characterized by aberrant glycosylation of alpha-dystroglycan (α-DG), collectively known as dystroglycanopathies. The clinical variations associated with FKRP mutations range from mild limb-girdle muscular dystrophy type 2I with predominantly muscle phenotypes to severe Walker-Warburg syndrome and muscle-eye-brain disease with striking structural brain and eye defects. In the present study, we have generated animal models and demonstrated that ablation of FKRP functions is embryonic lethal and that the homozygous-null embryos die before reaching E12.5. The homozygous knock-in mouse carrying the missense P448L mutation almost completely lacks functional glycosylation of α-DG in muscles and brain, validating the essential role of FKRP in the functional glycosylation of α-DG. However, the knock-in mouse survives and develops a wide range of structural abnormalities in the central nervous system, characteristics of neuronal migration defects. The brain and eye defects are highly reminiscent of the phenotypes seen in severe dystroglycanopathy patients. In addition, skeletal muscles develop progressive muscular dystrophy. Our results confirm that post-translational modifications of α-DG are essential for normal development of the brain and eyes. In addition, both the mutation itself and the levels of FKRP expression are equally critical for the survival of the animals. The exceptionally wide clinical spectrums recapitulated in the P448L mice also suggest the involvement of other factors in the disease progression. The mutant mouse represents a valuable model to further elucidate the functions of FKRP and develop therapies for FKRP-related muscular dystrophies.


Assuntos
Encéfalo/embriologia , Distroglicanas/metabolismo , Olho/embriologia , Desenvolvimento Muscular , Músculo Esquelético/embriologia , Distrofia Muscular Animal , Proteínas/metabolismo , Animais , Southern Blotting , Western Blotting , Encéfalo/fisiologia , Olho/metabolismo , Imunofluorescência , Deleção de Genes , Expressão Gênica , Técnicas de Introdução de Genes , Glicosilação , Malformações do Desenvolvimento Cortical do Grupo II , Camundongos , Camundongos Knockout , Modelos Animais , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , Proteínas/genética
9.
Biochim Biophys Acta ; 1802(2): 253-8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19900540

RESUMO

Mutations in the fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) as well as other severe muscle disorders, including Walker-Warburg syndrome, muscle-eye-brain disease, and congenital muscular dystrophy type 1C. The FKRP gene encodes a putative glycosyltransferase, but its precise localization and functions have yet to be determined. In the present study, we demonstrated that normal FKRP is secreted into culture medium and mutations alter the pattern of secretion in CHO cells. L276I mutation associated with mild disease phenotype was shown to reduce the level of secretion whereas P448L and C318Y mutations associated with severe disease phenotype almost abolished the secretion. However, a truncated FKRP mutant protein lacking the entire C-terminal 185 amino acids due to the E310X nonsense mutation was able to secrete as efficiently as the normal FKRP. The N-terminal signal peptide sequence is apparently cleaved from the secreted FKRP proteins. Alteration of the secretion pathway by different mutations and spontaneous read-through of nonsense mutation may contribute to wide variations in phenotypes associated with FKRP-related diseases.


Assuntos
Proteínas/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Western Blotting , Células CHO , Cardiomiopatias/genética , Cricetinae , Cricetulus , Amplificação de Genes , Humanos , Microssomos/metabolismo , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Distrofias Musculares/genética , Proteínas/metabolismo , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
11.
Artigo em Inglês | MEDLINE | ID: mdl-18211681

RESUMO

Detecting acute nerve compression using neurophysiologic studies is an important part of the practice of clinical intra-operative neurophysiology. The goal of this paper was to study the changes in the compound muscle action potential (CMAP) during acute mechanical compression. This is the type of injury most likely to occur during surgery. Thus, understanding the changes in the CMAP during this type of injury will be useful in the detection and prevention using intra-operative neurophysiologic monitoring. The model involved compression of the hamster sciatic nerve over a region of 1.3 mm with pressures up to 2000 mmHg for times on the order of 3 minutes. In this model CMAP amplitude dropped to 50% of its baseline value when a pressure of roughly 1000 mmHg is applied while, at the same time, nerve conduction velocities decline by only 5%. The ability to detect statistically significant changes in the CMAP at low force levels using other descriptors of the CMAP including duration, latency variation, etc alone or in conjunction with amplitude and velocity measures was investigated. However, these other parameters did not allow for earlier detection of significant changes. This study focused on a model in which nerve injury on a short time scale is purely mechanical in origin. It demonstrated that a pure compression injury produced large changes in CMAP amplitude prior to large changes in conduction velocity. On the other hand, ischemic and stretch injuries are associated with larger changes in conduction velocity for a given value of CMAP amplitude reduction.

12.
Exp Neurol ; 205(1): 257-69, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17397833

RESUMO

Sarcoglycans are originally identified in muscle for their involvement in limb-girdle muscular dystrophies. They form a multi-meric complex (alpha-, beta-, gamma-, delta-sarcoglycan) that associates with dystrophin, dystroglycan and other proteins to constitute the larger dystrophin-glycoprotein complex at the muscle membrane. Three sarcoglycan subunits (epsilon-, beta-, delta-sarcoglycan) were previously identified in Schwann cells and shown to associate with dystroglycan and a Schwann cell-specific dystrophin isoform (Dp116) at the outermost Schwann cell membrane. Currently, little is known about the exact composition and function of the sarcoglycan complex in the peripheral nervous system. In this study, we showed that the Schwann cell sarcoglycan complex consists of epsilon-, beta-, delta-sarcoglycan and the newly identified zeta-sarcoglycan subunit. The expression of sarcoglycans precedes the onset of myelination and is induced by neurons. In sarcoglycan-deficient BIO14.6 hamsters, loss of the Schwann cell sarcoglycan complex reduces the steady state levels of alpha-dystroglycan and Dp116. Ultrastructural analysis of sciatic nerves from the mutant animals revealed altered myelin sheaths and disorganized Schmidt-Lanterman incisures indicative of myelin instability. The disruption in myelin structure increased in severity with age. Nerve conduction studies also showed subtle electrophysiological abnormalities in the BIO14.6 hamsters consistent with reduced myelin stability. Together, these findings suggest an important role of sarcoglycans in the stability of peripheral nerve myelin.


Assuntos
Bainha de Mielina/química , Sarcoglicanas/fisiologia , Células de Schwann/metabolismo , Envelhecimento , Animais , Células Cultivadas , Técnicas de Cocultura , Cricetinae , Citoplasma/ultraestrutura , Estabilidade de Medicamentos , Distroglicanas/química , Distroglicanas/metabolismo , Eletrofisiologia , Masculino , Microscopia Eletrônica , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Sistema Nervoso/fisiopatologia , Condução Nervosa , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Sarcoglicanas/deficiência , Sarcoglicanas/metabolismo , Células de Schwann/ultraestrutura , Nervo Isquiático/ultraestrutura , Fatores de Tempo
13.
Biochim Biophys Acta ; 1772(5): 570-9, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17382524

RESUMO

The sarcoglycan complex in muscle consists of alpha-, beta-, gamma- and delta-sarcoglycan and is part of the larger dystrophin-glycoprotein complex (DGC), which is essential for maintaining muscle membrane integrity. Mutations in any of the four sarcoglycans cause limb-girdle muscular dystrophies (LGMD). In this report, we have identified a novel interaction between delta-sarcoglycan and the 16 kDa subunit c (16K) of vacuolar H(+)-ATPase. Co-expression studies in heterologous cell system revealed that 16K interacts specifically with delta-sarcoglycan and the highly related gamma-sarcoglycan through the transmembrane domains. In cultured C2C12 myotubes, 16K forms a complex with sarcoglycans at the plasma membrane. Loss of sarcoglycans in the sarcoglycan-deficient BIO14.6 hamster destabilizes the DGC and alters the localization of 16K at the sarcolemma. In addition, the steady state level of beta(1)-integrin is increased. Recent studies have shown that 16K also interacts directly with beta(1)-integrin and our data demonstrated that sarcoglycans, 16K and beta(1)-integrin were immunoprecipitated together in C2C12 myotubes. Since sarcoglycans have been proposed to participate in bi-directional signaling with integrins, our findings suggest that 16K might mediate the communication between sarcoglycans and integrins and play an important role in the pathogenesis of muscular dystrophy.


Assuntos
Sarcoglicanas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Cercopithecus aethiops , Cricetinae , Integrina beta1/metabolismo , Masculino , Camundongos , Microscopia Imunoeletrônica , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Sarcolema/metabolismo , Sarcolema/ultraestrutura , Transdução de Sinais
14.
Exp Cell Res ; 312(9): 1610-25, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16524571

RESUMO

Mutations in sarcoglycans have been reported to cause autosomal-recessive limb-girdle muscular dystrophies. In skeletal and cardiac muscle, sarcoglycans are assembled into a complex on the sarcolemma from four subunits (alpha, beta, gamma, delta). In this report, we present a detailed structural analysis of sarcoglycans using deletion study, limited proteolysis and co-immunoprecipitation. Our results indicate that the extracellular regions of sarcoglycans consist of distinctive functional domains connected by proteinase K-sensitive sites. The N-terminal half domains are required for sarcoglycan interaction. The C-terminal half domains of beta-, gamma- and delta-sarcoglycan consist of a cysteine-rich motif and a previously unrecognized conserved sequence, both of which are essential for plasma membrane localization. Using a heterologous expression system, we demonstrate that missense sarcoglycan mutations affect sarcoglycan complex assembly and/or localization to the cell surface. Our data suggest that the formation of a stable complex is necessary but not sufficient for plasma membrane targeting. Finally, we provide evidence that the beta/delta-sarcoglycan core can associate with the C-terminus of dystrophin. Our results therefore generate important information on the structure of the sarcoglycan complex and the molecular mechanisms underlying the effects of various sarcoglycan mutations in muscular dystrophies.


Assuntos
Membrana Celular/metabolismo , Sarcoglicanas/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Cercopithecus aethiops , Cisteína/genética , Distrofina/metabolismo , Glicosilação , Imunoprecipitação , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico/fisiologia , Ratos , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção
15.
J Appl Physiol (1985) ; 100(1): 212-20, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16195392

RESUMO

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From -70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased V(max) without appreciable changes in K(m) for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either alpha1- or alpha2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with alpha-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered V(max) but not K(m) of Na+-K+-ATPase in postinfarction rat myocytes.


Assuntos
Ativação do Canal Iônico , Proteínas de Membrana/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Regulação da Expressão Gênica , Masculino , Potenciais da Membrana , Proteínas de Membrana/genética , Fosfoproteínas/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Muscle Nerve ; 29(3): 409-19, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14981741

RESUMO

Mutations in sarcoglycans (SG) have been reported to cause autosomal-recessive limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy. In skeletal and cardiac muscle, sarcoglycans exist as a complex of four transmembrane proteins (alpha-, beta-, gamma-, and delta-SG). In this study, the assembly of the sarcoglycan complex was examined in a heterologous expression system. Our results demonstrated that the assembly process occurs as a discrete stepwise process. We found that beta-SG appears to play an initiating role and its association with delta-SG is essential for the proper localization of the sarcoglycan complex to the cell membrane. The incorporation of alpha-SG into the sarcoglycan complex occurs at the final stage by interaction with gamma-SG. These findings were supported by chemical cross-linking of endogenous sarcoglycans in cultured myotubes. We have also provided evidence that glycosylation-defective mutations in beta-SG and a common mutation in gamma-SG (C283Y) disrupt sarcoglycan-complex formation. Our proposed model for the assembly and structure of sarcoglycans should generate important insight into their function in muscle as well as their role in muscular dystrophies and cardiomyopathies.


Assuntos
Diferenciação Celular/genética , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Células COS , Cardiomiopatias/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/genética , Distroglicanas , Fibroblastos , Glicosilação , Substâncias Macromoleculares , Glicoproteínas de Membrana/genética , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/genética , Mutação/genética , Mioblastos , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Sarcoglicanas , Transfecção
17.
J Clin Invest ; 110(6): 807-14, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12235112

RESUMO

Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.


Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Adolescente , Animais , Biópsia , Células da Medula Óssea/ultraestrutura , Núcleo Celular/ultraestrutura , Criança , Distrofina/genética , Distrofina/metabolismo , Éxons/genética , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Lactente , Masculino , Músculo Esquelético/citologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Imunodeficiência Combinada Severa/terapia , Fatores de Tempo
18.
J Biol Chem ; 277(37): 34375-82, 2002 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-12167663

RESUMO

Mammalian isoforms of calcium-permeable Drosophila transient receptor potential channels (TRPC) are involved in the sustained phase of calcium entry in nonexcitable cells. Erythropoietin (Epo) stimulates a rise in intracellular calcium ([Ca](i)) via activation of voltage-independent calcium channel(s) in erythroid cells. Here, involvement of murine orthologs of classical TRPC in the Epo-modulated increase in [Ca](i) was examined. RT-PCR of TRPC 1-6 revealed high expression of only TRPC2 in Epo-dependent cell lines HCD-57 and Ba/F3 Epo-R, in which Epo stimulates a rise in [Ca](i). Using RT-PCR, Western blotting, and immunolocalization, expression of the longest isoform of mTRPC2, clone 14, was demonstrated in HCD-57 cells, Ba/F3 Epo-R cells, and primary murine erythroblasts. To determine whether erythropoietin is capable of modulating calcium influx through TRPC2, CHO cells were cotransfected with Epo-R subcloned into pTracer-CMV and either murine TRPC2 clone 14 or TRPC6, a negative control, into pQBI50. Successful transfection of Epo-R was verified in single cells by detection of green fluorescent protein from pTracer-CMV using digital video imaging, and successful transfection of TRPC was confirmed by detection of blue fluorescent protein fused through a flexible linker to TRPC. [Ca](i) changes were simultaneously monitored in cells loaded with Rhod-2 or Fura Red. Epo stimulation of CHO cells cotransfected with Epo-R and TRPC2 resulted in a rise in [Ca](i) above base line (372 +/- 71%), which was significantly greater (p < or = 0.0007) than that seen in cells transfected with TRPC6 or empty pQBI50 vector. This rise in [Ca](i) required Epo and extracellular calcium. These results identify a calcium-permeable channel, TRPC2, in erythroid cells and demonstrate modulation of calcium influx through this channel by erythropoietin.


Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Eritropoetina/farmacologia , Canais Iônicos , Proteínas de Membrana , Animais , Células CHO , Cricetinae , Camundongos , Camundongos Endogâmicos C57BL , Receptores da Eritropoetina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPM , Transfecção
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