RESUMEN
The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed among the five subspecies of Salmonella enterica, is found in many clinically relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, ß-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and ß-dystrobrevin. The interaction between SseM and ß-2-syntrophin and α-dystrobrevin was verified in Salmonella Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of ß-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A ΔsseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the ΔsseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence. IMPORTANCE: In Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein ß-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.
Asunto(s)
Proteínas Bacterianas , Salmonella enteritidis , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Humanos , Ratones , Virulencia , Salmonella enteritidis/genética , Salmonella enteritidis/metabolismo , Salmonella enteritidis/patogenicidad , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Infecciones por Salmonella/microbiología , Proteínas Asociadas a la Distrofina/metabolismo , Proteínas Asociadas a la Distrofina/genética , Islas Genómicas , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Proteómica , Modelos Animales de Enfermedad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genéticaRESUMEN
Neuromuscular junctions transmit signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibers and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process. Our proteomic screen identified several putative αDystrobrevin-1 interactors recruited to the Y730 site in phosphorylated and unphosphorylated states. Amongst various actin-modulating proteins, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology in cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis anterior. However, muscle strength examination did not reveal apparent deficits in knockout animals.
Asunto(s)
Cortactina , Proteínas Asociadas a la Distrofina , Ratones Noqueados , Unión Neuromuscular , Animales , Unión Neuromuscular/metabolismo , Cortactina/metabolismo , Cortactina/genética , Ratones , Proteínas Asociadas a la Distrofina/metabolismo , Proteínas Asociadas a la Distrofina/genética , Músculo Esquelético/metabolismo , Humanos , FosforilaciónRESUMEN
The dystrophin-glycoprotein complex (DGC) plays a crucial role in maintaining the structural integrity of the plasma membrane and the neuromuscular junction. In this study, we investigated the impact of the deficiency of α-dystrobrevin (αdbn), a component of the DGC, on the homeostasis of intracellular organelles, specifically mitochondria and the sarcoplasmic reticulum (SR). In αdbn deficient muscles, we observed a significant increase in the membrane-bound ATP synthase complex levels, a marker for mitochondria in oxidative muscle fiber types compared to wild-type. Furthermore, examination of muscle fibers deficient in αdbn using electron microscopy revealed profound alterations in the organization of mitochondria and the SR within certain myofibrils of muscle fibers. This included the formation of hyper-branched intermyofibrillar mitochondria with extended connections, an extensive network spanning several myofibrils, and a substantial increase in the number/density of subsarcolemmal mitochondria. Concurrently, in some cases, we observed significant structural alterations in mitochondria, such as cristae loss, fragmentation, swelling, and the formation of vacuoles and inclusions within the mitochondrial matrix cristae. Muscles deficient in αdbn also displayed notable alterations in the morphology of the SR, along with the formation of distinct anomalous concentric SR structures known as whorls. These whorls were prevalent in αdbn-deficient mice but were absent in wild-type muscles. These results suggest a crucial role of the DGC αdbn in regulating intracellular organelles, particularly mitochondria and the SR, within muscle cells. The remodeling of the SR and the formation of whorls may represent a novel mechanism of the unfolded protein response (UPR) in muscle cells.
Asunto(s)
Proteínas Asociadas a la Distrofina , Distrofina , Mitocondrias , Retículo Sarcoplasmático , Animales , Ratones , Distrofina/genética , Distrofina/metabolismo , Distrofina/deficiencia , Proteínas Asociadas a la Distrofina/genética , Proteínas Asociadas a la Distrofina/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas/genética , Glicoproteínas/deficiencia , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mitocondrias/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestructuraRESUMEN
PURPOSE: We report a case of Hermansky-Pudlak Syndrome type 7 (HPS-7) caused by a homozygous variant in the dystrobrevin-binding protein 1 gene (DTNBP1) and highlight the genetic challenges associated with this rare disorder. METHODS: Case report. Literature review was performed by searching PubMed on May 2023, without language or date restriction, using the following terms: Hermansky-Pudlak syndrome, Hermansky-Pudlak syndrome type 7, and dystrobrevin-binding protein 1 gene. RESULTS: We report a case of a 69-year-old Portuguese female who presented for ophthalmic evaluation with long-standing severe visual impairment, pronounced photophobia, right-eye esotropia, and bilateral pendular nystagmus. Anterior segment examination revealed iris transillumination defects, while the ocular fundus showed hypopigmentation and the absence of the foveal reflex. The patient had a history of oculocutaneous albinism (OCA) and recurrent epistaxis. Her family history was positive for first-degree consanguineous parents and a deceased sister at young age who also exhibited OCA and recurrent epistaxis. Genetic testing identified a homozygous pathogenic nonsense variant in the DTNBP1, c.307C>T p.(Gln103*). The patient's clinical features and genetic testing support the diagnosis of HPS-7. The identified variant has been previously reported in the literature, in adult patients of Portuguese descent. CONCLUSION: This work highlights the genetic complexity of HPS-7 and emphasizes the importance of genetic testing in the diagnosis of this rare disorder. The identification of a rare pathogenic variant expands our understanding of HPS-7 genetics and suggests a possible founder effect in the Portuguese population.
Asunto(s)
Síndrome de Hermanski-Pudlak , Humanos , Femenino , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/diagnóstico , Anciano , Proteínas Asociadas a la Distrofina/genética , Linaje , MutaciónRESUMEN
DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.
Asunto(s)
Trastorno del Espectro Autista , Distrofias Musculares , Neuropéptidos , Ratones , Humanos , Animales , Niño , Distrofina/genética , Distrofina/metabolismo , Trastorno del Espectro Autista/metabolismo , Distrofias Musculares/metabolismo , Distroglicanos/metabolismo , Empalme Alternativo , Músculo Esquelético/patología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Proteínas Asociadas a la Distrofina/genética , Proteínas Asociadas a la Distrofina/metabolismoRESUMEN
α-Dystrobrevin (α-DB) is a major component of the dystrophin-associated protein complex (DAPC). Knockout (KO) of α-DB in the brain is associated with astrocytic abnormalities and loss of neuronal GABA receptor clustering. Mutations in DAPC proteins are associated with altered dopamine signaling and cognitive and psychiatric disorders, including schizophrenia. This study tested the hypothesis that motivation and associated underlying biological pathways are altered in the absence of α-DB expression. Male wildtype and α-DB KO mice were tested for measures of motivation, executive function and extinction in the rodent touchscreen apparatus. Subsequently, brain tissues were evaluated for mRNA and/or protein levels of dysbindin-1, dopamine transporter and receptor 1 and 2, mu opioid receptor 1 (mOR1) and cannabinoid receptor 1 (CB1). α-DB KO mice had significantly increased motivation for the appetitive reward, while measures of executive function and extinction were unaffected. No differences were observed between wildtype and KO animals on mRNA levels of dysbindin-1 or any of the dopamine markers. mRNA levels of mOR1were significantly decreased in the caudate-putamen and nucleus accumbens of α-DB KO compared to WT animals, but protein levels were unaltered. However, CB1 protein levels were significantly increased in the prefrontal cortex and decreased in the nucleus accumbens of α-DB KO mice. Triple-labelling immunohistochemistry confirmed that changes in CB1 were not specific to astrocytes. These results highlight a novel role for α-DB in the regulation of appetitive motivation that may have implications for other behaviours that involve the dopaminergic and endocannabinoid systems.
Asunto(s)
Dopamina , Proteínas Asociadas a la Distrofina , Motivación , Receptores de Cannabinoides , Animales , Encéfalo/metabolismo , Dopamina/metabolismo , Disbindina/metabolismo , Proteínas Asociadas a la Distrofina/genética , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Receptores de Cannabinoides/genética , Receptores de Cannabinoides/metabolismo , RecompensaRESUMEN
Alzheimer's disease (AD) is a genetically complex disease for which nearly 40 loci have now been identified via genome-wide association studies (GWAS). We attempted to identify groups of rare variants (alternate allele frequency <0.01) associated with AD in a region-based, whole-genome sequencing (WGS) association study (rvGWAS) of two independent AD family datasets (NIMH/NIA; 2247 individuals; 605 families). Employing a sliding window approach across the genome, we identified several regions that achieved association p values <10-6, using the burden test or the SKAT statistic. The genomic region around the dystobrevin beta (DTNB) gene was identified with the burden and SKAT test and replicated in case/control samples from the ADSP study reaching genome-wide significance after meta-analysis (pmeta = 4.74 × 10-8). SKAT analysis also revealed region-based association around the Discs large homolog 2 (DLG2) gene and replicated in case/control samples from the ADSP study (pmeta = 1 × 10-6). In conclusion, in a region-based rvGWAS of AD we identified two novel AD genes, DLG2 and DTNB, based on association with rare variants.
Asunto(s)
Enfermedad de Alzheimer , Proteínas Asociadas a la Distrofina/genética , Neuropéptidos/genética , Enfermedad de Alzheimer/genética , Ácido Ditionitrobenzoico , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Genómica , Guanilato-Quinasas/genética , Humanos , Polimorfismo de Nucleótido Simple/genética , Proteínas Supresoras de Tumor/genética , Secuenciación Completa del GenomaRESUMEN
α-syntrophin (α-syn) and α-dystrobrevin (α-dbn), two components of the dystrophin-glycoprotein complex, are essential for the maturation and maintenance of the neuromuscular junction (NMJ) and mice deficient in either α-syn or α-dbn exhibit similar synaptic defects. However, the functional link between these two proteins and whether they exert distinct or redundant functions in the postsynaptic organization of the NMJ remain largely unknown. We generated and analyzed the synaptic phenotype of double heterozygote (α-dbn+/-, α-syn+/-), and double homozygote knockout (α-dbn-/-; α-syn-/-) mice and examined the ability of individual molecules to restore their defects in the synaptic phenotype. We showed that in double heterozygote mice, NMJs have normal synaptic phenotypes and no signs of muscular dystrophy. However, in double knockout mice (α-dbn-/-; α-syn-/-), the synaptic phenotype (the density, the turnover and the distribution of AChRs within synaptic branches) is more severely impaired than in single α-dbn-/- or α-syn-/- mutants. Furthermore, double mutant and single α-dbn-/- mutant mice showed more severe exercise-induced fatigue and more significant reductions in grip strength than single α-syn-/- mutant and wild-type. Finally, we showed that the overexpression of the transgene α-syn-GFP in muscles of double mutant restores primarily the abnormal extensions of membrane containing AChRs that extend beyond synaptic gutters and lack synaptic folds, whereas the overexpression of α-dbn essentially restores the abnormal dispersion of patchy AChR aggregates in the crests of synaptic folds. Altogether, these data suggest that α-syn and α-dbn act in parallel pathways and exert distinct functions on the postsynaptic structural organization of NMJs.
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Distrofina , Receptores Colinérgicos , Animales , Proteínas de Unión al Calcio , Distrofina/genética , Distrofina/metabolismo , Proteínas Asociadas a la Distrofina/genética , Proteínas Asociadas a la Distrofina/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
Atrial fibrillation (AF) is a morbid and heritable irregular cardiac rhythm that affects about 2%-3% of the population. Patients with early-onset AF have a strong genetic association with the disease; nonetheless, the exact underlying mechanisms need clarification. We herein present our evaluation of a 2-generation Iranian pedigree with early-onset AF. Whole-exome sequencing was applied to elucidate the genetic predisposition. Direct DNA sequencing was utilized to confirm and screen the variants in the proband and his available family members. The pathogenicity of the identified nucleotide variations was scrutinized via either segregation analysis in the family or in silico predictive software. The comprehensive variant analysis revealed a missense variant (c.G681C, p.E227D, rs1477078144) in the human α-dystrobrevin gene (DTNA), which is rare in genetic databases. Most in silico analyses have predicted this variant as a disease-causing variant, and the variant is co-segregated with the disease phenotype in the family. Previous studies have demonstrated the association between the DTNA gene and left ventricular noncompaction cardiomyopathy. Taken together, we provide the first evidence of an association between a nucleotide variation in the DTNA gene and early-onset AF in an Iranian family. However, the genetic testing of AF in the Iranian population is still limited. This finding not only further confirms the significant role of genetics in the incidence of early-onset AF but also expands the spectrum of the gene variations that lead to AF. Additionally, it may have further implications for the treatment and prevention of AF.
Asunto(s)
Fibrilación Atrial/genética , Análisis Mutacional de ADN , Proteínas Asociadas a la Distrofina/genética , Secuenciación del Exoma , Frecuencia Cardíaca/genética , Mutación Missense , Neuropéptidos/genética , Adolescente , Fibrilación Atrial/diagnóstico , Fibrilación Atrial/fisiopatología , Electrocardiografía , Femenino , Predisposición Genética a la Enfermedad , Herencia , Humanos , Irán , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Valor Predictivo de las Pruebas , Adulto JovenRESUMEN
BACKGROUND: Numerous approaches have been proposed for the detection of epistatic interactions within GWAS datasets in order to better understand the drivers of disease and genetics. METHODS: A selection of state-of-the-art approaches were assessed. These included the statistical tests, fast-epistasis, BOOST, logistic regression and wtest; swarm intelligence methods, namely AntEpiSeeker, epiACO and CINOEDV; and data mining approaches, including MDR, GSS, SNPRuler and MPI3SNP. Data were simulated to provide randomly generated models with no individual main effects at different heritabilities (pure epistasis) as well as models based on penetrance tables with some main effects (impure epistasis). Detection of both two and three locus interactions were assessed across a total of 1,560 simulated datasets. The different methods were also applied to a section of the UK biobank cohort for Atrial Fibrillation. RESULTS: For pure, two locus interactions, PLINK's implementation of BOOST recovered the highest number of correct interactions, with 53.9% and significantly better performing than the other methods (p = 4.52e - 36). For impure two locus interactions, MDR exhibited the best performance, recovering 62.2% of the most significant impure epistatic interactions (p = 6.31e - 90 for all but one test). The assessment of three locus interaction prediction revealed that wtest recovered the highest number (17.2%) of pure epistatic interactions(p = 8.49e - 14). wtest also recovered the highest number of three locus impure epistatic interactions (p = 6.76e - 48) while AntEpiSeeker ranked as the most significant the highest number of such interactions (40.5%). Finally, when applied to a real dataset for Atrial Fibrillation, most notably finding an interaction between SYNE2 and DTNB.
Asunto(s)
Fibrilación Atrial/genética , Epistasis Genética , Sitios Genéticos , Modelos Genéticos , Penetrancia , Algoritmos , Alelos , Minería de Datos/métodos , Proteínas Asociadas a la Distrofina/genética , Frecuencia de los Genes , Estudio de Asociación del Genoma Completo/métodos , Genotipo , Humanos , Modelos Lineales , Proteínas de Microfilamentos/genética , Reducción de Dimensionalidad Multifactorial , Proteínas del Tejido Nervioso/genética , Neuropéptidos/genética , Polimorfismo de Nucleótido Simple , Curva ROCRESUMEN
Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA-tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71, although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinß (Dtnb), whereas glia-associated Dp71 did with Dg and dystrobrevinα (Dtna). Thus, our data indicate that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.
Asunto(s)
Distroglicanos/metabolismo , Proteínas Asociadas a la Distrofina/metabolismo , Distrofina/metabolismo , Neuroglía/metabolismo , Neuropéptidos/metabolismo , Sinapsis/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Distroglicanos/genética , Distrofina/genética , Proteínas Asociadas a la Distrofina/genética , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Ratones Transgénicos , Microscopía Confocal , Neuronas/metabolismo , Neuropéptidos/genética , Unión ProteicaRESUMEN
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.
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Distrofina/genética , Exones , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/genética , Animales , Animales Modificados Genéticamente , Dependovirus/genética , Modelos Animales de Enfermedad , Proteínas Asociadas a la Distrofina/genética , Proteínas Asociadas a la Distrofina/metabolismo , Femenino , Eliminación de Gen , Masculino , Fibras Musculares Esqueléticas/patología , Técnicas de Transferencia Nuclear , Oligonucleótidos Antisentido/genética , Sarcolema/metabolismo , Porcinos , Porcinos EnanosRESUMEN
RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas Asociadas a la Distrofina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Tiorredoxinas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Sitios de Unión , Clonación Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Asociadas a la Distrofina/química , Proteínas Asociadas a la Distrofina/genética , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Moleculares , Ovario/citología , Ovario/metabolismo , Poli U/química , Poli U/genética , Poli U/metabolismo , Unión Proteica , ARN/química , ARN/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/química , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Sudden unexplained death (SUD) takes up a considerable part in overall sudden death cases, especially in adolescents and young adults. During the past decade, many channelopathy- and cardiomyopathy-associated single nucleotide variants (SNVs) have been identified in SUD studies by means of postmortem molecular autopsy, yet the number of cases that remain inconclusive is still high. Recent studies had suggested that structural variants (SVs) might play an important role in SUD, but there is no consensus on the impact of SVs on inherited cardiac diseases. In this study, we searched for potentially pathogenic SVs in 244 genes associated with cardiac diseases. Whole-exome sequencing and appropriate data analysis were performed in 45 SUD cases. Re-analysis of the exome data according to the current ACMG guidelines identified 14 pathogenic or likely pathogenic variants in 10 (22.2%) out of the 45 SUD cases, whereof 2 (4.4%) individuals had variants with likely functional effects in the channelopathy-associated genes SCN5A and TRDN and 1 (2.2%) individual in the cardiomyopathy-associated gene DTNA. In addition, 18 structural variants (SVs) were identified in 15 out of the 45 individuals. Two SVs with likely functional impairment were found in the coding regions of PDSS2 and TRPM4 in 2 SUD cases (4.4%). Both were identified as heterozygous deletions, which were confirmed by multiplex ligation-dependent probe amplification. In conclusion, our findings support that SVs could contribute to the pathology of the sudden death event in some of the cases and therefore should be investigated on a routine basis in suspected SUD cases.
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Muerte Súbita/patología , Variación Estructural del Genoma/genética , Cardiopatías/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Transferasas Alquil y Aril , Proteínas Portadoras/genética , Niño , Preescolar , Estudios de Cohortes , Proteínas Asociadas a la Distrofina/genética , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Proteínas Musculares/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Neuropéptidos/genética , Sistemas de Lectura Abierta , Suiza/epidemiología , Canales Catiónicos TRPM , Secuenciación del ExomaRESUMEN
Aquaporin-4 (AQP4) water channels and gap junction proteins (connexins) are two classes of astrocytic membrane proteins critically involved in brain water and ion homeostasis. AQP4 channels are anchored by α1-syntrophin to the perivascular astrocytic endfoot membrane domains where they control water flux at the blood-brain interface while connexins cluster at the lateral aspects of the astrocytic endfeet forming gap junctions that allow water and ions to dissipate through the astrocyte syncytium. Recent studies have pointed to an interdependence between astrocytic AQP4 and astrocytic gap junctions but the underlying mechanism remains to be explored. Here we use a novel transgenic mouse line to unravel whether ß1-syntrophin (coexpressed with α1-syntrophin in astrocytic plasma membranes) is implicated in the expression of AQP4 isoforms and formation of gap junctions in brain. Our results show that while the effect of ß1-syntrophin deletion is rather limited, double knockout of α1- and ß1-syntrophin causes a downregulation of the novel AQP4 isoform AQP4ex and an increase in the number of astrocytic gap junctions. The present study highlight the importance of syntrophins in orchestrating specialized functional domains of brain astrocytes.
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Acuaporina 4/genética , Encéfalo/metabolismo , Proteínas de Unión al Calcio/genética , Conexina 43/genética , Proteínas Asociadas a la Distrofina/genética , Proteínas de la Membrana/genética , Proteínas Musculares/genética , Animales , Membrana Celular/genética , Uniones Comunicantes/genética , Regulación de la Expresión Génica/genética , Homeostasis/genética , Iones/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos/genética , Ratones Transgénicos/metabolismo , Agua/metabolismoRESUMEN
Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.
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Movimiento Celular , Diacilglicerol Quinasa/fisiología , Proteínas Asociadas a la Distrofina/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Neuropéptidos/metabolismo , Proteína Quinasa C-alfa/farmacología , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Diglicéridos/metabolismo , Proteínas Asociadas a la Distrofina/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Neuropéptidos/genética , Dominios Proteicos , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
PURPOSE: To investigate the associations of Single Nucleotide Polymorphisms (SNPs) in the SNTB1 gene with high myopia in a Han Chinese population. MATERIALS AND METHODS: Based on previous studies, four SNPs from the SNTB1 gene were chosen for genotyping. This is a case-control genetic association study comprising 193 high myopia participants and 135 normal emmetropic controls from a Han Chinese population. Allelic frequencies of the SNPs and haplotypes were compared to assess the associations of the SNPs with high myopia and axial length (AL). RESULTS: The SNPs rs7839488 (effect allele: A; OR = 0.685), rs4395927 (effect allele: T; OR = 0.692), and rs6469937 (effect allele: A; OR = 0.683) displayed significant associations with high myopia initially (P = .044, 0.049, and 0.035, respectively), but did not withstand permutation testing (all Ppermutation>0.05). rs6469937 displayed associations with high myopia in the dominant model (AG+AA: OR = 0.609) against GG (reference). rs6469937 was also associated with AL in the dominant model (AG+AA: Beta = -0.58) against GG (reference). The haplotype analysis demonstrated ATGA as the protective haplotype against high myopia, which remained statistically significant in permutation testing (Ppermutation = 0.045). CONCLUSIONS: Our findings are suggestive that SNTB1 is associated with high myopia in a Han Chinese population.
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Pueblo Asiatico/genética , Proteínas Asociadas a la Distrofina/genética , Miopía Degenerativa/genética , Polimorfismo de Nucleótido Simple/genética , Estudios de Casos y Controles , China/epidemiología , Frecuencia de los Genes , Estudios de Asociación Genética , Técnicas de Genotipaje , Haplotipos , Humanos , Masculino , Adulto JovenRESUMEN
BACKGROUND: Esophageal cancer (EC) ranks the eighth in morbidity and the sixth in mortality around the whole world, which is an aggressive malignancy. To authenticate potential therapeutic targets for EC is therefore imperative. Although miR-301b might display changed expression in esophageal adenocarcinoma by utilizing Taqman miRNA profiling analysis, much less is known about the impact of miR-301b in EC. METHODS AND RESULTS: By analyzing the data of 187 cancer tissues and 13 normal samples from TCGA database, we discovered that miR-301b was highly expressed in EC tissues. Then, RT-qPCR determined that miR-301b was up-regulated in EC cell lines (ECA109, JAR, TE-1 and OE33). Besides, miR-301b expression level was higher in ESCC cell line-TE-1 cells and lower in ESCC cell line-ECA109 cells compared to other EC cell lines. Hence, ECA109 cell line was used to up-regulate miR-301b expression while TE-1 cell line was applied to down-regulate miR-301b expression in the subsequent experiments. Additionally, OE33, as an ECA cell line, was applied to upregulate miR-301b expression to reflect the influence of miR-301b overexpression on EC progression. More interestingly, miR-301b appeared to act as a promoting effect on the proliferation of EC cells, which was tested by CCK8. Dystrobrevin alpha (DTNA) was a targeting gene of miR-301b, which was predicted by the websites of miRanda, miRWalk and TargetScan. Additionally, DTNA was low expressed in EC tissues and was an independent predictor of EC. Meanwhile, the low expression of DTNA was related to worse overall survival in EC patients. The Pearson correlation coefficient analyzed that DTNA expression was negatively correlated with miR-301b. Furthermore, RT-qPCR and western blotting assays ulteriorly indicated that DTNA was negatively modulated by miR-301b. The facilitating impact of miR-301b re-expression on ECA109 and OE33 cell growth, invasion and migration was receded by DTNA over-expression, whilst the repressive effect of miR-301b ablation on TE-1 cell growth, invasion and migration was inversed by DTNA silencing. Overexpression of miR-301b accelerated EC cell growth, migration and invasion through targeting DTNA. CONCLUSIONS: Above all, we concluded that miR-301b was concerned with the progression of EC via regulating DTNA, suggesting that miR-301b and its target gene, DTNA, might serve as predictive biomarkers for EC therapy.
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Proteínas Asociadas a la Distrofina , Neoplasias Esofágicas , MicroARNs , Neuropéptidos , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular/genética , Proteínas Asociadas a la Distrofina/genética , Proteínas Asociadas a la Distrofina/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica , Neuropéptidos/genética , Neuropéptidos/metabolismoRESUMEN
The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap-α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap-α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Asociadas a la Distrofina/metabolismo , Complejos Multiproteicos/metabolismo , Neuropéptidos/metabolismo , Receptores Colinérgicos/biosíntesis , Proteínas de Anclaje a la Quinasa A/genética , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Asociadas a la Distrofina/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Complejos Multiproteicos/genética , Neuropéptidos/genética , Dominios Proteicos , Estabilidad Proteica , Receptores Colinérgicos/genéticaRESUMEN
We report the case of a 23-year-old woman with a not yet described (to the best of our knowledge) association of left ventricle non-compaction with both atrial and ventricular defects. Family genetic survey concluded to, a probably sporadic, E101K gene mutation.