RESUMEN
Viral protein R (Vpr) is a small accessory protein of 96 amino acids that is present in Human and simian immunodeficiency viruses. Among the very different properties that Vpr possesses we can find cell penetrating capabilities. Based on this and on its capacity to interact with nucleic acids we previously investigated the DNA transfection properties of Vpr and subfragments thereof. We found that fragments of the C-terminal helical domain of Vpr are able to deliver efficiently plasmid DNA into different cell lines. As the amphipathic helix may play a role in the interactions with membranes, we investigated whether insertion of a proline residue in the α-helix modifies the transfection properties of Vpr. Unexpectedly, we found that the resulting Vpr55-82 Pro70 peptide was even more efficient than wild type Vpr55-82 in the gene delivery assays. Using circular dichroism, light scattering and solid-state NMR techniques, we characterized the secondary structure and interactions of Vpr and several mutants with model membranes. A model is proposed where the proline shifts the dissociation equilibrium of the peptide-cargo complex and thereby its endosomal release.
Asunto(s)
Péptidos de Penetración Celular/química , Técnicas de Transferencia de Gen , Membrana Dobles de Lípidos/química , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/química , Sustitución de Aminoácidos , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/metabolismo , Células HEK293 , VIH-1/química , Humanos , Isoleucina/química , Isoleucina/genética , Prolina/química , Prolina/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Recessive mutations in PYROXD1, encoding an oxidoreductase, were recently reported in families with congenital myopathy or limb-girdle muscular dystrophy. Here we describe three novel PYROXD1 families at the clinical, histological, and genetic level. Histological analyses on muscle biopsies from all families revealed fiber size variability, endomysial fibrosis, and muscle fibers with multiple internal nuclei and cores. Further characterization of the structural muscle defects uncovered aggregations of myofibrillar proteins, and provided evidence for enhanced oxidative stress. Sequencing identified homozygous or compound heterozygous PYROXD1 mutations including the first deep intronic mutation reinforcing a cryptic donor splice site and resulting in mRNA instability through exonisation of an intronic segment. Overall, this work expands the PYROXD1 mutation spectrum, defines and specifies the histopathological hallmarks of the disorder, and indicates that oxidative stress contributes to the pathomechanism. Comparison of all new and published cases uncovered a genotype/phenotype correlation with a more severe and early-onset phenotypic presentation of patients harboring splice mutations resulting in reduced PYROXD1 protein levels compared with patients carrying missense mutations.
Asunto(s)
Enfermedades Musculares/diagnóstico , Enfermedades Musculares/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Adulto , Anciano , Secuencia de Bases , Niño , Femenino , Humanos , Recién Nacido , Masculino , Enfermedades Musculares/patologíaRESUMEN
Muscle contraction requires specialized membrane structures with precise geometry and relies on the concerted interplay of electrical stimulation and Ca2+ release, known as excitation-contraction coupling (ECC). The membrane structure hosting ECC is called triad in skeletal muscle and dyad in cardiac muscle, and structural or functional defects of triads and dyads have been observed in a variety of myopathies and cardiomyopathies. Based on their function, the proteins localized at the triad/dyad can be classified into three molecular pathways: the Ca2+ release complex (CRC), store-operated Ca2+ entry (SOCE), and membrane remodeling. All three are mechanistically linked, and consequently, aberrations in any of these pathways cause similar disease entities. This review provides an overview of the clinical and genetic spectrum of triad and dyad defects with a main focus of attention on the underlying pathomechanisms.
Asunto(s)
Calcio/metabolismo , Cardiomiopatías/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Enfermedades Musculares/metabolismo , Animales , Cardiomiopatías/genética , Homeostasis , Humanos , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Miocardio/metabolismoRESUMEN
Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Miotonía Congénita/genética , Miotonía Congénita/metabolismo , Adolescente , Adulto , Calcio/metabolismo , Canales de Calcio Tipo L , Células Cultivadas , Niño , Estudios de Cohortes , Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Musculares/metabolismo , Células Musculares/patología , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación , Miotonía Congénita/diagnóstico por imagen , Miotonía Congénita/patología , Fenotipo , Homología de Secuencia de Aminoácido , Adulto JovenRESUMEN
This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.