RESUMEN
Adipocytes release immune mediators that contribute to diabetes-associated inflammatory processes. As the stress protein heat shock protein 60 (Hsp60) induces proinflammatory adipocyte activities, we hypothesized that adipocytes of diabetes-predisposed mice exhibit an increased proinflammatory reactivity to Hsp60. Preadipocytes and mature adipocytes from nonobese diabetic (NOD), New Zealand obese (NZO), and C57BL/6J mice were analyzed for Hsp60 binding, Hsp60-activated signaling pathways, and Hsp60-induced release of the chemokine CXCL-1 (KC), interleukin 6 (IL-6), and macrophage chemoattractant protein-1 (MCP-1). Hsp60 showed specific binding to (pre-)adipocytes of NOD, NZO, and C57BL/6J mice. Hsp60 binding involved conserved binding structure(s) and Hsp60 epitopes and was strongest to NZO mouse-derived mature adipocytes. Hsp60 exposure induced KC, IL-6, and MCP-1 release from (pre-)adipocytes of all mouse strains with a pronounced increase of IL-6 release from NZO mouse-derived adipocytes. Compared to NOD and C57BL/6J mouse derived cells, Hsp60-induced formation of IL-6, KC, and MCP-1 from NZO mouse-derived (pre-)adipocytes strongly depended on NF κ B-activation. Increased Hsp60 binding and Hsp60-induced IL-6 release by mature adipocytes of NZO mice suggest that enhanced adipocyte reactivity to the stress signal Hsp60 contributes to inflammatory processes underlying diabetes associated with obesity and insulin resistance.
Asunto(s)
Adipocitos/metabolismo , Chaperonina 60/metabolismo , Citocinas/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Mitocondriales/metabolismo , Obesidad/metabolismo , Paniculitis/metabolismo , Regulación hacia Arriba , Adipocitos/inmunología , Adipocitos/patología , Adipogénesis , Animales , Células Cultivadas , Chaperonina 60/genética , Quimiocina CCL2/agonistas , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/agonistas , Quimiocina CXCL1/biosíntesis , Quimiocina CXCL1/metabolismo , Citocinas/agonistas , Citocinas/biosíntesis , Femenino , Interleucina-6/agonistas , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Grasa Intraabdominal/inmunología , Grasa Intraabdominal/metabolismo , Grasa Intraabdominal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Obesos , Proteínas Mitocondriales/genética , FN-kappa B/agonistas , FN-kappa B/metabolismo , Obesidad/inmunología , Obesidad/patología , Paniculitis/inmunología , Paniculitis/patología , Proteínas Recombinantes/metabolismoRESUMEN
The human Usher syndrome (USH) is a complex ciliopathy with at least 12 chromosomal loci assigned to three clinical subtypes, USH1-3. The heterogeneous USH proteins are organized into protein networks. Here, we identified Magi2 (membrane-associated guanylate kinase inverted-2) as a new component of the USH protein interactome, binding to the multifunctional scaffold protein SANS (USH1G). We showed that the SANS-Magi2 complex assembly is regulated by the phosphorylation of an internal PDZ-binding motif in the sterile alpha motif domain of SANS by the protein kinase CK2. We affirmed Magi2's role in receptor-mediated, clathrin-dependent endocytosis and showed that phosphorylated SANS tightly regulates Magi2-mediated endocytosis. Specific depletions by RNAi revealed that SANS and Magi2-mediated endocytosis regulates aspects of ciliogenesis. Furthermore, we demonstrated the localization of the SANS-Magi2 complex in the periciliary membrane complex facing the ciliary pocket of retinal photoreceptor cells in situ. Our data suggest that endocytotic processes may not only contribute to photoreceptor cell homeostasis but also counterbalance the periciliary membrane delivery accompanying the exocytosis processes for the cargo vesicle delivery. In USH1G patients, mutations in SANS eliminate Magi2 binding and thereby deregulate endocytosis, lead to defective ciliary transport modules and ultimately disrupt photoreceptor cell function inducing retinal degeneration.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Síndromes de Usher/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Clatrina/genética , Clatrina/metabolismo , Endocitosis , Regulación de la Expresión Génica , Guanilato-Quinasas , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Fosforilación , Células Fotorreceptoras de Vertebrados/patología , Cultivo Primario de Células , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Transducción de Señal , Síndromes de Usher/metabolismo , Síndromes de Usher/patologíaRESUMEN
The stress protein heat shock protein 60 (Hsp60) induces secretion of proinflammatory mediators from murine adipocytes. This study aimed to study Hsp60 as a mediator of adipose tissue inflammation and skeletal muscle cell (SkMC) insulin sensitivity and to quantify plasma Hsp60 concentrations in lean and obese individuals. Regulation of Hsp60 release and Hsp60-induced cytokine secretion and signaling was measured in human adipocytes and SkMCs. Adipocytes exhibited higher Hsp60 release than preadipocytes and SkMCs, which was further stimulated by cytokines and Toll-like receptor (TLR)-4 activation. Hsp60 activated extracellular signal-related kinase (ERK)-1/2, Jun NH(2)-terminal kinase (JNK), p38, nuclear factor (NF)-κB, and impaired insulin-stimulated Akt phosphorylation in adipocytes. Furthermore, Hsp60 stimulated adipocytes to secrete tumor necrosis factor-α, interleukin (IL)-6, and IL-8. In SkMCs, Hsp60 activated ERK1/2, JNK, and NF-κB and inhibits insulin signaling and insulin-stimulated glucose uptake. SkMCs released IL-6, IL-8, and monocyte chemoattractant protein-1 on Hsp60 stimulation. Plasma Hsp60 was higher in obese males than in lean males and correlated positively with BMI, blood pressure, leptin, and homeostasis model assessment-insulin resistance. In summary, Hsp60 is released by human adipocytes, increased in plasma of obese humans, and induces insulin resistance. This is accompanied by activation of proinflammatory signaling in human adipocytes and SkMCs. Thus, Hsp60 might be a factor underlying adipose tissue inflammation and obesity-associated metabolic disorders.
Asunto(s)
Tejido Adiposo/metabolismo , Chaperonina 60/fisiología , Inflamación/etiología , Resistencia a la Insulina , Adipocitos/metabolismo , Adulto , Células Cultivadas , Chaperonina 60/sangre , Quimiocina CCL2/metabolismo , Glucosa/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/metabolismoRESUMEN
The human Usher syndrome (USH) is the most frequent cause of combined hereditary deaf-blindness. USH is genetically heterogeneous with at least 11 chromosomal loci assigned to 3 clinical types, USH1-3. We have previously demonstrated that all USH1 and 2 proteins in the eye and the inner ear are organized into protein networks by scaffold proteins. This has contributed essentially to our current understanding of the function of USH proteins and explains why defects in proteins of different families cause very similar phenotypes. We have previously shown that the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) contributes to the periciliary protein network in retinal photoreceptor cells. This study aimed to further elucidate the role of SANS by identifying novel interaction partners. In yeast two-hybrid screens of retinal cDNA libraries we identified 30 novel putative interacting proteins binding to the central domain of SANS (CENT). We confirmed the direct binding of the phosphodiesterase 4D interacting protein (PDE4DIP), a Golgi associated protein synonymously named myomegalin, to the CENT domain of SANS by independent assays. Correlative immunohistochemical and electron microscopic analyses showed a co-localization of SANS and myomegalin in mammalian photoreceptor cells in close association with microtubules. Based on the present results we propose a role of the SANS-myomegalin complex in microtubule-dependent inner segment cargo transport towards the ciliary base of photoreceptor cells.
Asunto(s)
Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Retina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Bovinos , Células Cultivadas , Chlorocebus aethiops , Proteínas del Citoesqueleto , Humanos , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Unión Proteica/fisiología , Levaduras/genética , Levaduras/metabolismoRESUMEN
Adipocyte-derived mediators contribute to chronic, diabetes-associated inflammation. We recently demonstrated, that heat shock protein 60 (Hsp60) is an effective inductor of inflammatory adipocyte activities. In the present study, we characterized the initial Hsp60 binding to adipocyte receptor structures. Analyses with preadipocytes and adipocytes from the murine 3T3-L1 line and with primary cultures from the New Zealand obese mouse, a model of human obesity, revealed comparable specific, dose-dependent and saturable Hsp60 binding, confirming the characteristics of a ligand-receptor interaction. Furthermore, we identified the N-terminal regions aa1-50 and aa91-110 of the Hsp60 molecule as relevant epitopes involved in binding to receptor structures on these cells. Our results demonstrate differentiation-independent conserved Hsp60 reactivity in permanent and primary adipocytes, strongly indicating that Hsp60 is an important regulator of inflammatory adipocyte activities.
Asunto(s)
Adipocitos/metabolismo , Chaperonina 60/metabolismo , Células 3T3-L1 , Animales , Chaperonina 60/genética , Endocitosis , Humanos , Ligandos , Ratones , Ratones EndogámicosRESUMEN
PURPOSE: Usher syndrome is the most common form of hereditary deaf-blindness. It is both clinically and genetically heterogeneous. The USH2D protein whirlin interacts via its PDZ domains with other Usher-associated proteins containing a C-terminal type I PDZ-binding motif. These proteins co-localize with whirlin at the region of the connecting cilium and at the synapse of photoreceptor cells. This study was undertaken to identify novel, Usher syndrome-associated, interacting partners of whirlin and thereby obtain more insights into the function of whirlin. METHODS: The database of ciliary proteins was searched for proteins that are present in both the retina and inner ear and contain a PDZ-binding motif. Interactions with whirlin were evaluated by yeast two-hybrid analyses and validated by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization in the retina with immunofluorescence and immunoelectron microscopy. RESULTS: The L-type calcium channel subunit Ca(v)1.3 (alpha(1D)) specifically interacts with whirlin. In adult photoreceptors, Ca(v)1.3 (alpha(1D)) and whirlin co-localize in the region of the connecting cilium and at the synapse. During murine embryonic development, the expression patterns of the Whrn and Cacna1d genes show significant overlap and include expression in the eye, the inner ear, and the central nervous system. CONCLUSIONS: The findings indicate that Ca(v)1.3 (alpha(1D)) is connected to the Usher protein network. This conclusion leads to the hypothesis that, in the retina, whirlin scaffolds Ca(v)1.3 (alpha(1D)) and therefore contributes to the organization of calcium channels in the photoreceptor cells, where both proteins may be involved in membrane fusions.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Proteínas de la Membrana/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Animales , Western Blotting , Células COS , Canales de Calcio Tipo L/genética , Chlorocebus aethiops , Biología Computacional , Bases de Datos de Proteínas , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Cilio Conector de los Fotorreceptores/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Retina/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Adipocytes play important roles in lipid metabolism but also in the control of inflammatory processes. Based on our previous findings of heat shock protein (Hsp) 60-induced activation of preadipocytes we investigated whether the capacity of heat shock protein 60 (Hsp60) to interact with adipocytes and to stimulate their proinflammatory activity is determined by the differentiation state of the cells. Hsp60 bound to adipocytes and stimulated the release of inflammatory mediators independent of their differentiation state. Hsp60-adipocyte interactions revealed basic characteristics of a receptor-mediated process. Our findings characterize Hsp60 binding and Hsp60-induced release of proinflammatory mediators as fundamental properties of adipocytes independent of their differentiation state.
Asunto(s)
Adipocitos/inmunología , Adipocitos/fisiología , Diferenciación Celular/fisiología , Chaperonina 60/metabolismo , Mediadores de Inflamación/metabolismo , Células 3T3-L1 , Adipocitos/citología , Animales , Humanos , Ratones , Transducción de Señal/inmunologíaRESUMEN
A homozygous reciprocal translocation, 46,XY,t(10;11),t(10;11), was detected in a boy with non-syndromic congenital sensorineural hearing impairment. Both parents and their four other children were heterozygous translocation carriers, 46,XX,t(10;11) and 46,XY,t(10;11), respectively. Fluorescence in situ hybridization of region-specific clones to patient chromosomes was used to localize the breakpoints within bacterial artificial chromosome (BAC) RP11-108L7 on chromosome 10q24.3 and within BAC CTD-2527F12 on chromosome 11q23.3. Junction fragments were cloned by vector ligation and sequenced. The chromosome 10 breakpoint was identified within the PDZ domain containing 7 (PDZD7) gene, disrupting the open reading frame of transcript PDZD7-C (without PDZ domain) and the 5'-untranslated region of transcript PDZD7-D (with one PDZ and two prolin-rich domains). The chromosome 11 breakpoint was localized in an intergenic segment. Reverse transcriptase-polymerase chain reaction analysis revealed PDZD7 expression in the human inner ear. A murine Pdzd7 transcript that is most similar in structure to human PDZD7-D is known to be expressed in the adult inner ear and retina. PDZD7 shares sequence homology with the PDZ domain-containing genes, USH1C (harmonin) and DFNB31 (whirlin). Allelic mutations in harmonin and whirlin can cause both Usher syndrome (USH1C and USH2D, respectively) and congenital hearing impairment (DFNB18 and DFNB31, respectively). Protein-protein interaction assays revealed the integration of PDZD7 in the protein network related to the human Usher syndrome. Collectively, our data provide strong evidence that PDZD7 is a new autosomal-recessive deafness-causing gene and also a prime candidate gene for Usher syndrome.
Asunto(s)
Consanguinidad , Pérdida Auditiva/genética , Translocación Genética , Síndromes de Usher/genética , Secuencia de Aminoácidos , Secuencia de Bases , Preescolar , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 11/genética , Oído Interno/metabolismo , Femenino , Reordenamiento Génico , Pérdida Auditiva/congénito , Pérdida Auditiva/metabolismo , Heterocigoto , Homocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Síndromes de Usher/metabolismoRESUMEN
Usher syndrome (USH) and Leber congenital amaurosis (LCA) are autosomal recessive disorders resulting in syndromic and non-syndromic forms of blindness. In order to gain insight into the pathogenic mechanisms underlying retinal degeneration, we searched for interacting proteins of USH2A isoform B (USH2A(isoB)) and the LCA5-encoded protein lebercilin. We identified a novel isoform of the centrosomal ninein-like protein, hereby named Nlp isoform B (Nlp(isoB)), as a common interactor. Although we identified the capacity of this protein to bind calcium with one of its three EF-hand domains, the interacton with USH2A(isoB) did not depend on this. Upon expression in ARPE-19 cells, recombinant Nlp(isoB), lebercilin and USH2A(isoB) were all found to co-localize at the centrosomes. Staining of retinal sections with specific antibodies against all three proteins revealed their co-localization at the basal bodies of the photoreceptor-connecting cilia. Based on this subcellular localization and the nature of their previously identified binding partners, we hypothesize that the pathogenic mechanisms for LCA and USH show significant overlap and involve defects in ciliogenesis, cilia maintenance and intraflagellar and/or microtubule-based transport. The direct association of Nlp(isoB) with USH2A(isoB) and lebercilin indicates that Nlp can be considered as a novel candidate gene for USH, LCA and allied retinal ciliopathies.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Atrofia Óptica Hereditaria de Leber/metabolismo , Síndromes de Usher/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Atrofia Óptica Hereditaria de Leber/genética , Células Fotorreceptoras/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Wistar , Retina/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos , Síndromes de Usher/genéticaRESUMEN
The highly ordered distribution of neurons is an essential feature of a functional mammalian retina. Disruptions in the apico-basal polarity complexes at the outer limiting membrane (OLM) of the retina are associated with retinal patterning defects in vertebrates. We have analyzed the binding repertoire of MPP5/Pals1, a key member of the apico-basal Crumbs polarity complex, that has functionally conserved counterparts in zebrafish (nagie oko) and Drosophila (Stardust). We show that MPP5 interacts with its MAGUK family member MPP1/p55 at the OLM. Mechanistically, this interaction involves heterodimerization of both MAGUK modules in a directional fashion. MPP1 expression in the retina throughout development resembles the expression of whirlin, a multi-PDZ scaffold protein and an important organizer in the Usher protein network. We demonstrate that both proteins interact strongly by both a classical PDZ domain-to-PDZ binding motif (PBM) mechanism, and a mechanism involving internal epitopes. MPP1 and whirlin colocalize in the retina at the OLM, at the outer synaptic layer and at the basal bodies and the ciliary axoneme. In view of the known roles of the Crumbs and Usher protein networks, our findings suggest a novel link of the core developmental processes of actin polymerization and establishment/maintenance of apico-basal cell polarity through MPP1. These processes, essential in neural development and patterning of the retina, may be disrupted in eye disorders that are associated with defects in these protein networks.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas del Ojo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Retina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Sanguíneas/genética , Membrana Celular/metabolismo , Embrión de Mamíferos/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Ojo/genética , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Nucleósido-Fosfato Quinasa/química , Nucleósido-Fosfato Quinasa/genética , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Técnicas del Sistema de Dos HíbridosRESUMEN
Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-Løken syndrome (SLSN) or Joubert syndrome (JBTS). However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedades Cerebelosas/genética , Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Oftalmopatías/genética , Enfermedades Renales/genética , Proteínas/genética , Proteínas/metabolismo , Adulto , Animales , Línea Celular , Proteínas del Citoesqueleto , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Ratas , SíndromeRESUMEN
Usher syndrome is the most common form of deaf-blindness. The syndrome is both clinically and genetically heterogeneous, and to date, eight causative genes have been identified. The proteins encoded by these genes are part of a dynamic protein complex that is present in hair cells of the inner ear and in photoreceptor cells of the retina. The localization of the Usher proteins and the phenotype in animal models indicate that the Usher protein complex is essential in the morphogenesis of the stereocilia bundle in hair cells and in the calycal processes of photoreceptor cells. In addition, the Usher proteins are important in the synaptic processes of both cell types. The association of other proteins with the complex indicates functional links to a number of basic cell-biological processes. Prominently present is the connection to the dynamics of the actin cytoskeleton, involved in cellular morphology, cell polarity and cell-cell interactions. The Usher protein complex can also be linked to the cadherins/catenins in the adherens junction-associated protein complexes, suggesting a role in cell polarity and tissue organization. A third link can be established to the integrin transmembrane signaling network. The Usher interactome, as outlined in this review, participates in pathways common in inner ear and retina that are disrupted in the Usher syndrome.
Asunto(s)
Síndromes de Usher/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Oído Interno/metabolismo , Células Ciliadas Auditivas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Complejos Multiproteicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Retina/metabolismo , Sinapsis/metabolismo , Síndromes de Usher/etiología , Síndromes de Usher/metabolismoRESUMEN
Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. It is clinically and genetically heterogeneous and at least 12 chromosomal loci are assigned to three clinical USH types, namely USH1A-G, USH2A-C, USH3A (Davenport, S.L.H., Omenn, G.S., 1977. The heterogeneity of Usher syndrome. Vth Int. Conf. Birth Defects, Montreal; Petit, C., 2001. Usher syndrome: from genetics to pathogenesis. Annu. Rev. Genomics Hum. Genet. 2, 271-297). Mutations in USH type 1 genes cause the most severe form of USH. In USH1 patients, congenital deafness is combined with a pre-pubertal onset of retinitis pigmentosa (RP) and severe vestibular dysfunctions. Those with USH2 have moderate to severe congenital hearing loss, non-vestibular dysfunction and a later onset of RP. USH3 is characterized by variable RP and vestibular dysfunction combined with progressive hearing loss. The gene products of eight identified USH genes belong to different protein classes and families. There are five known USH1 molecules: the molecular motor myosin VIIa (USH1B); the two cell-cell adhesion cadherin proteins, cadherin 23 (USH1D) and protocadherin 15, (USH1F) and the scaffold proteins, harmonin (USH1C) and SANS (USH1G). In addition, two USH2 genes and one USH3A gene have been identified. The two USH2 genes code for the transmembrane protein USH2A, also termed USH2A ("usherin") and the G-protein-coupled 7-transmembrane receptor VLGR1b (USH2C), respectively, whereas the USH3A gene encodes clarin-1, a member of the clarin family which exhibits 4-transmembrane domains. Molecular analysis of USH1 protein function revealed that all five USH1 proteins are integrated into a protein network via binding to PDZ domains in the USH1C protein harmonin. Furthermore, this scaffold function of harmonin is supported by the USH1G protein SANS. Recently, we have shown that the USH2 proteins USH2A and VLGR1b as well as the candidate for USH2B, the sodium bicarbonate co-transporter NBC3, are also integrated into this USH protein network. In the inner ear, these interactions are essential for the differentiation of hair cell stereocilia but may also participate in the mechano-electrical signal transduction and the synaptic function of maturated hair cells. In the retina, the co-expression of all USH1 and USH2 proteins at the synapse of photoreceptor cells indicates that they are organized in an USH protein network there. The identification of the USH protein network indicates a common pathophysiological pathway in USH. Dysfunction or absence of any of the molecules in the mutual "interactome" related to the USH disease may lead to disruption of the network causing senso-neuronal degeneration in the inner ear and the retina, the clinical symptoms of USH.
Asunto(s)
Síndromes de Usher/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Sordera/congénito , Sordera/genética , Modelos Animales de Enfermedad , Dineínas/genética , Dineínas/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismo , Modelos Genéticos , Modelos Moleculares , Mutación/genética , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Degeneración Retiniana/genética , Síndromes de Usher/metabolismo , Vestíbulo del Laberinto/fisiopatologíaRESUMEN
Mutations in the DFNB31 gene encoding the PDZ scaffold protein whirlin are causative for hearing loss in man and mouse. Whirlin is known to be essential for the elongation process of the stereocilia of sensory hair cells in the inner ear, though its complete spatial and temporal expression patterns remained elusive. Here, we demonstrate that, in embryonic development, the gene is not only expressed in the inner ear, but also in the developing brain and the retina. Various isoforms of whirlin are widely and differentially expressed, and we provide evidence that whirlin directly associates with USH2A isoform b and VLGR1b, two proteins that we previously reported to be part of the Usher protein interactome. These proteins co-localize with whirlin at the synaptic regions of both photoreceptor cells and outer hair cells in the cochlea. These findings indicate that whirlin is part of a macromolecular PDZ protein scaffold that functions in the organization of the pre- and/or postsynaptic side of photoreceptor and hair cell synapses. Whirlin might be involved in synaptic adhesion through interaction with USH2A and VLGR1b as well as in synaptic development as suggested by its spatial and temporal expression patterns. In addition, we demonstrate that whirlin, USH2A and Vlgr1b co-localize at the connecting cilium and the outer limiting membrane of photoreceptor cells and in spiral ganglion neurons of the inner ear. Our data show that whirlin is connected to the dynamic Usher protein interactome and indicate that whirlin has a pleiotropic function in both the retina and the inner ear.
Asunto(s)
Cóclea/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismo , Animales , Células COS , Chlorocebus aethiops , Proteínas de la Matriz Extracelular/genética , Cobayas , Inmunohistoquímica , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. USH is clinically and genetically heterogeneous with at least 11 chromosomal loci assigned to the three USH types (USH1A-G, USH2A-C, USH3A). Although the different USH types exhibit almost the same phenotype in human, the identified USH genes encode for proteins which belong to very different protein classes and families. We and others recently reported that the scaffold protein harmonin (USH1C-gene product) integrates all identified USH1 molecules in a USH1-protein network. Here, we investigated the relationship between the USH2 molecules and this USH1-protein network. We show a molecular interaction between the scaffold protein harmonin (USH1C) and the USH2A protein, VLGR1 (USH2C) and the candidate for USH2B, NBC3. We pinpoint these interactions to interactions between the PDZ1 domain of harmonin and the PDZ-binding motifs at the C-termini of the USH2 proteins and NBC3. We demonstrate that USH2A, VLGR1 and NBC3 are co-expressed with the USH1-protein harmonin in the synaptic terminals of both retinal photoreceptors and inner ear hair cells. In hair cells, these USH proteins are also localized in the signal uptaking stereocilia. Our data indicate that the USH2 proteins and NBC3 are further partners in the supramolecular USH-protein network in the retina and inner ear which shed new light on the function of USH2 proteins and the entire USH-protein network. These findings provide first evidence for a molecular linkage between the pathophysiology in USH1 and USH2. The organization of USH molecules in a mutual 'interactome' related to the disease can explain the common phenotype in USH.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Síndromes de Usher/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Proteínas de la Matriz Extracelular/genética , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras/metabolismo , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Síndromes de Usher/fisiopatologíaRESUMEN
PURPOSE: The human Usher syndrome (USH) is the most common form of deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal onset of retinitis pigmentosa. Five corresponding genes of the seven USH1 genes have been cloned over the years. Recent studies indicated that three USH1 proteins, namely myosin VIIa (USH1B), SANS (USH1G), and cadherin 23 (USH1D) interact with the USH1C gene product harmonin. In these protein-protein complexes harmonin acts as the scaffold protein binding these USH1 molecules via its PDZ domains. The aim of the present study was to analyze whether or not the fifth identified USH1 protein protocadherin 15 (Pcdh15) also binds to harmonin and where these putative protein complexes might be localized in mammalian rod and cone photoreceptor cells. METHODS: In vitro binding assays (GST pull-down, yeast two-hybrid assay) were applied. Antibodies against bacterial expressed USH1 proteins were generated. Affinity purified antibodies were used in immunoblot analyses of brain fractions and isolated retinas, in immunofluorescence studies, and in immunoelectron microscopic studies of rodent retinas. RESULTS: We showed that Pcdh15 (USH1F) interacted with harmonin PDZ2. Immunocytochemistry revealed that Pcdh15 is expressed in photoreceptor cells of the mammalian retina, where it is colocalized with harmonin, myosin VIIa, and cadherin 23 at the synaptic terminal. Colocalization of Pcdh15 with harmonin was found at the base of the photoreceptor outer segment, where newly synthesized disk membranes are present. CONCLUSIONS: Our data indicate that harmonin-Pcdh15 interactions probably play a role in disk morphogenesis. Furthermore, we provide evidence that a complex composed of all USH1 molecules may assemble at the photoreceptor synapse. This USH protein complex can contribute to the cortical cytoskeletal matrices of the pre- and postsynaptic regions, which are thought to play a fundamental role in the structural and functional organization of the synaptic junction. Defects in any of the USH1-complex partners may result in photoreceptor dysfunction causing retinitis pigmentosa, the clinical phenotype in the retina of USH1 patients.