RESUMEN
Presbycusis, or age-related hearing loss (ARHL), is a major public health issue. About half the phenotypic variance has been attributed to genetic factors. Here, we assessed the contribution to presbycusis of ultrarare pathogenic variants, considered indicative of Mendelian forms. We focused on severe presbycusis without environmental or comorbidity risk factors and studied multiplex family age-related hearing loss (mARHL) and simplex/sporadic age-related hearing loss (sARHL) cases and controls with normal hearing by whole-exome sequencing. Ultrarare variants (allele frequency [AF] < 0.0001) of 35 genes responsible for autosomal dominant early-onset forms of deafness, predicted to be pathogenic, were detected in 25.7% of mARHL and 22.7% of sARHL cases vs. 7.5% of controls (P = 0.001); half were previously unknown (AF < 0.000002). MYO6, MYO7A, PTPRQ, and TECTA variants were present in 8.9% of ARHL cases but less than 1% of controls. Evidence for a causal role of variants in presbycusis was provided by pathogenicity prediction programs, documented haploinsufficiency, three-dimensional structure/function analyses, cell biology experiments, and reported early effects. We also established Tmc1N321I/+ mice, carrying the TMC1:p.(Asn327Ile) variant detected in an mARHL case, as a mouse model for a monogenic form of presbycusis. Deafness gene variants can thus result in a continuum of auditory phenotypes. Our findings demonstrate that the genetics of presbycusis is shaped by not only well-studied polygenic risk factors of small effect size revealed by common variants but also, ultrarare variants likely resulting in monogenic forms, thereby paving the way for treatment with emerging inner ear gene therapy.
Asunto(s)
Sordera/genética , Genes Dominantes , Mutación/genética , Presbiacusia/genética , Factores de Edad , Edad de Inicio , Animales , Estudios de Casos y Controles , Estudios de Cohortes , Heterocigoto , Humanos , Proteínas de la Membrana/genética , Ratones , MicroARNs/genética , Mitocondrias/genética , Secuenciación del ExomaRESUMEN
The mechanotransducer channels of auditory hair cells are gated by tip-links, oblique filaments that interconnect the stereocilia of the hair bundle. Tip-links stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. They are made of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study we address the role of sans, a putative scaffold protein and product of the USH1G gene. In Ush1g(-/-) mice, the cohesion of stereocilia is disrupted, and both the amplitude and the sensitivity of the transduction currents are reduced. In Ush1g(fl/fl)Myo15-cre(+/-) mice, the loss of sans occurs postnatally and the stereocilia remain cohesive. In these mice, there is a decrease in the amplitude of the total transducer current with no loss in sensitivity, and the tips of the stereocilia in the short and middle rows lose their prolate shape, features that can be attributed to the loss of tip-links. Furthermore, stereocilia from these rows undergo a dramatic reduction in length, suggesting that the mechanotransduction machinery has a positive effect on F-actin polymerization. Sans interacts with the cytoplasmic domains of cadherin-23 and protocadherin-15 in vitro and is absent from the hair bundle in mice defective for either of the two cadherins. Because sans localizes mainly to the tips of short- and middle-row stereocilia in vivo, we conclude that it belongs to a molecular complex at the lower end of the tip-link and plays a critical role in the maintenance of this link.
Asunto(s)
Actinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/fisiología , Análisis de Varianza , Animales , Proteínas Relacionadas con las Cadherinas , Cadherinas/metabolismo , Cilios/metabolismo , Electrofisiología , Técnica del Anticuerpo Fluorescente , Vectores Genéticos/genética , Células Ciliadas Auditivas/ultraestructura , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Proteínas del Tejido Nervioso/genética , Polimerizacion , Precursores de Proteínas/metabolismo , Transducción de Señal/genéticaRESUMEN
Auditory neuropathy spectrum disorder (ANSD) refers to a range of hearing impairments characterized by an impaired transmission of sound from the cochlea to the brain. This defect can be due to a lesion or defect in the inner hair cell (IHC), IHC ribbon synapse (e.g., pre-synaptic release of glutamate), postsynaptic terminals of the spiral ganglion neurons, or demyelination and axonal loss within the auditory nerve. To date, the only clinical treatment options for ANSD are hearing aids and cochlear implantation. However, despite the advances in hearing-aid and cochlear-implant technologies, the quality of perceived sound still cannot match that of the normal ear. Recent advanced genetic diagnostics and clinical audiology made it possible to identify the precise site of a lesion and to characterize the specific disease mechanisms of ANSD, thus bringing renewed hope to the treatment or prevention of auditory neurodegeneration. Moreover, genetic routes involving the replacement or corrective editing of mutant sequences or defected genes to repair damaged cells for the future restoration of hearing in deaf people are showing promise. In this review, we provide an update on recent discoveries in the molecular pathophysiology of genetic lesions, auditory synaptopathy and neuropathy, and gene-therapy research towards hearing restoration in rodent models and in clinical trials.
RESUMEN
Cadherin-23 is a component of early transient lateral links of the auditory sensory cells' hair bundle, the mechanoreceptive structure to sound. This protein also makes up the upper part of the tip links that control gating of the mechanoelectrical transduction channels. We addressed the issue of the molecular complex that anchors these links to the hair bundle F-actin core. By using surface plasmon resonance assays, we show that the cytoplasmic regions of the two cadherin-23 isoforms that do or do not contain the exon68-encoded peptide directly interact with harmonin, a submembrane PDZ (post-synaptic density, disc large, zonula occludens) domain-containing protein, with unusually high affinity. This interaction involves the harmonin Nter-PDZ1 supramodule, but not the C-terminal PDZ-binding motif of cadherin-23. We establish that cadherin-23 directly binds to the tail of myosin VIIa. Moreover, cadherin-23, harmonin and myosin VIIa can form a ternary complex, which suggests that myosin VIIa applies tension forces on hair bundle links. We also show that the cadherin-23 cytoplasmic region, harmonin and myosin VIIa interact with phospholipids on synthetic liposomes. Harmonin and the cytoplasmic region of cadherin-23, both independently and as a binary complex, can bind specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), which may account for the role of this phospholipid in the adaptation of mechanoelectrical transduction in the hair bundle. The distributions of cadherin-23, harmonin, myosin VIIa and PI(4,5)P(2) in the growing and mature auditory hair bundles as well as the abnormal locations of harmonin and myosin VIIa in cadherin-23 null mutant mice strongly support the functional relevance of these interactions.
Asunto(s)
Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Miosinas/metabolismo , Fosfolípidos/metabolismo , Síndromes de Usher/metabolismo , Animales , Cadherinas/química , Cadherinas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Femenino , Células Ciliadas Auditivas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Miosina VIIa , Miosinas/química , Miosinas/genética , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Síndromes de Usher/genéticaRESUMEN
The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.
Asunto(s)
Sordera/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Sinapsis/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Sordera/genética , Modelos Animales de Enfermedad , Fenómenos Electrofisiológicos , Endocitosis , Femenino , Células Ciliadas Auditivas Internas/química , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cadenas Pesadas de Miosina/genética , Sinapsis/químicaRESUMEN
Here we solve a 2.4-A structure of a truncated version of the reverse-direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulin molecules. The structure reveals only minor differences in the motor domain from that in plus-end directed myosins, with the exception of two unique inserts. The first is near the nucleotide-binding pocket and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a novel target motif within the insert. This serves to redirect the effective 'lever arm' of myosin VI, which includes a second calmodulin bound to an 'IQ motif', towards the pointed (minus) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to provide a full explanation of the movements of myosin VI.
Asunto(s)
Movimiento , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/metabolismo , Actinas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calmodulina/metabolismo , Cristalografía por Rayos X , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-Actividad , PorcinosRESUMEN
BACKGROUND: Pathogenic variants of USH1C, encoding a PDZ-domain-containing protein called harmonin, have been known to cause autosomal recessive syndromic or nonsyndromic hearing loss (NSHL). We identified a causative gene in a large Korean family with NSHL showing a typical pattern of autosomal dominant (AD) inheritance. METHODS: Exome sequencing was performed for five affected and three unaffected individuals in this family. Following identification of a candidate gene variant, segregation analysis and functional studies, including circular dichroism and biolayer interferometry experiments, were performed. RESULTS: A novel USH1C heterozygous missense variant (c.667G>T;p.Gly223Cys) was shown to segregate with the NSHL phenotype in this family. This variant affects an amino acid residue located in the highly conserved carboxylate-binding loop of the harmonin PDZ2 domain and is predicted to disturb the interaction with cadherin-related 23 (cdh23). The affinity of the variant PDZ2 domain for a biotinylated synthetic peptide containing the PDZ-binding motif of cdh23 was approximately 16-fold lower than that of the wild-type PDZ2 domain and that this inaccessibility of the binding site was caused by a conformational change in the variant PDZ2 domain. CONCLUSIONS: A heterozygous variant of USH1C that interferes with the interaction between cdh23 and harmonin causes novel AD-NSHL.
Asunto(s)
Cadherinas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas del Citoesqueleto/genética , Sordera/patología , Adulto , Anciano , Proteínas Relacionadas con las Cadherinas , Análisis Mutacional de ADN , Sordera/genética , Femenino , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Dominios PDZ , Linaje , Unión ProteicaRESUMEN
The hair bundle of cochlear hair cells is the site of auditory mechanoelectrical transduction. It is formed by three rows of stiff microvilli-like protrusions of graduated heights, the short, middle-sized, and tall stereocilia. In developing and mature sensory hair cells, stereocilia are connected to each other by various types of fibrous links. Two unconventional cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23), form the tip-links, whose tension gates the hair cell mechanoelectrical transduction channels. These proteins also form transient lateral links connecting neighboring stereocilia during hair bundle morphogenesis. The proteins involved in anchoring these diverse links to the stereocilia dense actin cytoskeleton remain largely unknown. We show that the long isoform of whirlin (L-whirlin), a PDZ domain-containing submembrane scaffold protein, is present at the tips of the tall stereocilia in mature hair cells, together with PCDH15 isoforms CD1 and CD2; L-whirlin localization to the ankle-link region in developing hair bundles moreover depends on the presence of PCDH15-CD1 also localizing there. We further demonstrate that L-whirlin binds to PCDH15 and CDH23 with moderate-to-high affinities in vitro. From these results, we suggest that L-whirlin is part of the molecular complexes bridging PCDH15-, and possibly CDH23-containing lateral links to the cytoskeleton in immature and mature stereocilia.
Asunto(s)
Cadherinas/metabolismo , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas de la Membrana/metabolismo , Precursores de Proteínas/metabolismo , Animales , Proteínas Relacionadas con las Cadherinas , Diferenciación Celular/fisiología , Femenino , Masculino , Mecanotransducción Celular/fisiología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo/métodos , Isoformas de Proteínas/metabolismo , Estereocilios/metabolismoRESUMEN
Hearing is a mechanical and neurochemical process, which occurs in the hair cells of inner ear that converts the sound vibrations into electrical signals transmitted to the brain. The multi-PDZ scaffolding protein whirlin plays a critical role in the formation and function of stereocilia exposed at the surface of hair cells. In this article, we reported seven stereociliary proteins that encode PDZ binding motifs (PBM) and interact with whirlin PDZ3, where four of them are first reported. We solved the atomic resolution structures of complexes between whirlin PDZ3 and the PBMs of myosin 15a, CASK, harmonin a1 and taperin. Interestingly, the PBM of CASK and taperin are rare non-canonical PBM, which are not localized at the extreme C terminus. This large capacity to accommodate various partners could be related to the distinct functions of whirlin at different stages of the hair cell development.
Asunto(s)
Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Dominios PDZ/fisiología , Unión Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Guanilato-Quinasas/metabolismo , Humanos , Miosinas/metabolismo , Proteínas , Estereocilios/metabolismoRESUMEN
Several lines of evidence indicate that very large G-protein-coupled receptor 1 (Vlgr1) makes up the ankle links that connect the stereocilia of hair cells at their base. Here, we show that the transmembrane protein usherin, the putative transmembrane protein vezatin, and the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing submembrane protein whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells and are absent in Vlgr1-/- mice that lack the ankle links. Direct in vitro interactions between these four proteins further support their involvement in a molecular complex associated with the ankle links and scaffolded by whirlin. In addition, the delocalization of these proteins in myosin VIIa defective mutant mice as well as the myosin VIIa tail direct interactions with vezatin, whirlin, and, we show, Vlgr1 and usherin, suggest that myosin VIIa conveys proteins of the ankle-link complex to the stereocilia. Adenylyl cyclase 6, which was found at the base of stereocilia, was both overexpressed and mislocated in Vlgr1-/- mice. In postnatal day 7 Vlgr1-/- mice, mechanoelectrical transduction currents evoked by displacements of the hair bundle toward the tallest stereocilia (i.e., in the excitatory direction) were reduced in outer but not inner hair cells. In both cell types, stimulation of the hair bundle in the opposite direction paradoxically resulted in significant transduction currents. The absence of ankle-link-mediated cohesive forces within hair bundles lacking Vlgr1 may account for the electrophysiological results. However, because some long cadherin-23 isoforms could no longer be detected in Vlgr1-/- mice shortly after birth, the loss of some apical links could be involved too. The premature disappearance of these cadherin isoforms in the Vlgr1-/- mutant argues in favor of a signaling function of the ankle links in hair bundle differentiation.
Asunto(s)
Cóclea/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Ciliadas Auditivas/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Animales Recién Nacidos , Proteínas Portadoras/metabolismo , Quelantes/farmacología , Cilios/fisiología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Embrión de Mamíferos , Proteínas de la Matriz Extracelular/metabolismo , Células Ciliadas Auditivas/ultraestructura , Mecanotransducción Celular/genética , Mecanotransducción Celular/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo/métodos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Receptores Acoplados a Proteínas G/deficiencia , Subtilisina/farmacologíaRESUMEN
Mutations in the gene encoding harmonin, a multi-PDZ domain-containing submembrane protein, cause Usher syndrome type 1 (congenital deafness and balance disorder, and early-onset sight loss). The structure of the protein and biological activities of its three different classes of splice isoforms (a, b, and c) remain poorly understood. Combining biochemical and biophysical analyses, we show that harmonin-a1 can switch between open and closed conformations through intramolecular binding of its C-terminal PDZ-binding motif to its N-terminal supramodule NTD-PDZ1 and through a flexible PDZ2-PDZ3 linker. This conformational switch presumably extends to most harmonin isoforms, and it is expected to have an impact on the interaction with some binding partners, as shown here for cadherin-related 23, another component of the hair cell mechanoelectrical transduction machinery.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Cadherinas/metabolismo , Proteínas de Ciclo Celular , Dicroismo Circular , Proteínas del Citoesqueleto , Células HEK293 , Humanos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dominios Proteicos , Dispersión del Ángulo Pequeño , Transducción de Señal , Resonancia por Plasmón de Superficie , Difracción de Rayos XRESUMEN
Hearing relies on the transduction of sound-evoked vibrations into electric signals, occurring in the stereocilia bundle of hair cells. The bundle is organized in a staircase pattern formed by rows of packed stereocilia. This architecture is pivotal to transduction and involves a network of scaffolding proteins with hitherto uncharacterized features. Key interactions in this network are mediated by PDZ domains. Here, we describe the architecture of the first two PDZ domains of whirlin, a protein involved in these assemblies and associated with congenital deaf-blindness. C-terminal hairpin extensions of the PDZ domains mediate the transient supramodular assembly, which improves the binding capacity of the first domain. We determined a detailed structural model of the closed conformation of the PDZ tandem and characterized its equilibrium with an ensemble of open conformations. The structural and dynamic behavior of this PDZ tandem provides key insights into the regulatory mechanisms involved in the hearing machinery.
Asunto(s)
Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Dominios PDZ , Péptidos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Péptidos/síntesis química , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
Defects of CIB2, calcium- and integrin-binding protein 2, have been reported to cause isolated deafness, DFNB48 and Usher syndrome type-IJ, characterized by congenital profound deafness, balance defects and blindness. We report here two new nonsense mutations (pGln12* and pTyr110*) in CIB2 patients displaying nonsyndromic profound hearing loss, with no evidence of vestibular or retinal dysfunction. Also, the generated CIB2-/- mice display an early onset profound deafness and have normal balance and retinal functions. In these mice, the mechanoelectrical transduction currents are totally abolished in the auditory hair cells, whilst they remain unchanged in the vestibular hair cells. The hair bundle morphological abnormalities of CIB2-/- mice, unlike those of mice defective for the other five known USH1 proteins, begin only after birth and lead to regression of the stereocilia and rapid hair-cell death. This essential role of CIB2 in mechanotransduction and cell survival that, we show, is restricted to the cochlea, probably accounts for the presence in CIB2-/- mice and CIB2 patients, unlike in Usher syndrome, of isolated hearing loss without balance and vision deficits.
Asunto(s)
Proteínas de Unión al Calcio/genética , Sordera/diagnóstico , Células Ciliadas Auditivas Internas/metabolismo , Mecanotransducción Celular/fisiología , Animales , Umbral Auditivo , Conducta Animal , Proteínas de Unión al Calcio/deficiencia , Supervivencia Celular , Sordera/genética , Modelos Animales de Enfermedad , Ojo/diagnóstico por imagen , Ojo/patología , Femenino , Células Ciliadas Auditivas Internas/patología , Humanos , Masculino , Aprendizaje por Laberinto , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linaje , Polimorfismo de Nucleótido Simple , Retina/patología , Retina/fisiologíaRESUMEN
Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (OtofAla515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.
Asunto(s)
Células Ciliadas Auditivas/fisiología , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Receptores Sensibles al Calcio/metabolismo , Sinapsis/fisiología , Vesículas Sinápticas/metabolismo , Animales , Calcio/metabolismo , Técnicas de Sustitución del Gen , Proteínas de la Membrana/genética , Ratones , Unión Proteica , Receptores Sensibles al Calcio/genéticaRESUMEN
Usher syndrome (USH) is an autosomal recessive disorder characterized by a dual sensory impairment affecting hearing and vision. USH is clinically and genetically heterogeneous. Ten different causal genes have been reported. We studied the molecular bases of the disease in 18 unrelated Algerian patients by targeted-exome sequencing, and identified the causal biallelic mutations in all of them: 16 patients carried the mutations at the homozygous state and 2 at the compound heterozygous state. Nine of the 17 different mutations detected in MYO7A (1 of 5 mutations), CDH23 (4 of 7 mutations), PCDH15 (1 mutation), USH1C (1 mutation), USH1G (1 mutation), and USH2A (1 of 2 mutations), had not been previously reported. The deleterious consequences of a missense mutation of CDH23 (p.Asp1501Asn) and the in-frame single codon deletion in USH1G (p.Ala397del) on the corresponding proteins were predicted from the solved 3D-structures of extracellular cadherin (EC) domains of cadherin-23 and the sterile alpha motif (SAM) domain of USH1G/sans, respectively. In addition, we were able to show that the USH1G mutation is likely to affect the binding interface between the SAM domain and USH1C/harmonin. This should spur the use of 3D-structures, not only of isolated protein domains, but also of protein-protein interaction interfaces, to predict the functional impact of mutations detected in the USH genes.
Asunto(s)
Predisposición Genética a la Enfermedad , Variación Genética , Síndromes de Usher/genética , Argelia , Humanos , Mutación MissenseRESUMEN
Among the myosin superfamily, myosin VI differs from all others by a reverse directionality and a particular motility. Little structural information is available for myosin VI. It is known that it binds one calmodulin (CaM) by means of a single "IQ motif" and that myosin VI contains a specific insert located at the junction between the motor domain (MD) and the lever arm, likely to play a critical role for the unusual motility previously observed. Electrospray ionization mass spectrometry (MS) was used to determine the CaM and Ca2+ stoichiometries in several myosin VI constructs. In particular, the experimental conditions required for the observation of multiprotein/Ca2+ noncovalent assemblies are detailed for two truncated MD constructs (less than 20 kDa) and for three full MD constructs (more than 90 KDa). The specificity of the detected stoichiometries is discussed for each construct and the resolving power of Time of Flight mass spectrometry is stressed, in particular for the detection of metal ions binding to high molecular weight complexes. MS reveals a new CaM binding site for myosin VI and highlights a different behavior for the five myosin VI constructs versus Ca2+ binding. In addition to these stoichiometry based experiments, gas-phase dissociation analyses on intact complexes are described. They reveal that Ca2+ transfer between protein partners occurs during the dissociation process for one construct with a full MD. Charge-transfer and dissociation behavior has allowed to draw structural assumptions for the interaction of the MD with the CaM N-terminal lobe.
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Calmodulina/metabolismo , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Animales , Sitios de Unión , ADN Complementario , Mutagénesis , Cadenas Pesadas de Miosina/genética , Estructura Terciaria de Proteína , PorcinosRESUMEN
Protocadherin-15 (Pcdh15) is a component of the tip-links, the extracellular filaments that gate hair cell mechano-electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano-electrical transduction during hair-bundle development, but whether any of these isoforms composes the tip-link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post-natal hair cell-specific conditional knockout mice (Pcdh15ex38-fl/ex38-fl Myo15-cre+/-) that lose only this isoform after normal hair-bundle development, we show that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15-CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano-electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects.
Asunto(s)
Cadherinas/genética , Sordera/genética , Células Ciliadas Auditivas/citología , Precursores de Proteínas/genética , Animales , Proteínas Relacionadas con las Cadherinas , Cadherinas/análisis , Niño , Femenino , Mutación del Sistema de Lectura , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestructura , Humanos , Masculino , Mecanotransducción Celular , Ratones , Ratones Noqueados , Mutación , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Precursores de Proteínas/análisisRESUMEN
We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααß-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.
Asunto(s)
Anticuerpos Antiidiotipos/química , Cristalografía por Rayos X , Hematoporfirinas/química , Peroxidasas , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/inmunología , Sitios de Unión/inmunología , Sitios de Unión de Anticuerpos/inmunología , Hematoporfirinas/inmunología , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Estructura Molecular , Peroxidasas/química , Peroxidasas/inmunología , Peroxidasas/metabolismo , Conformación ProteicaRESUMEN
The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins-myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans-do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner-outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.