RESUMO
Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).
Assuntos
Síndromes de Usher , Humanos , Síndromes de Usher/genética , Síndromes de Usher/terapia , Síndromes de Usher/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Retina/metabolismo , Células Fotorreceptoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome-the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.
Assuntos
Processamento Alternativo/genética , Proteínas do Tecido Nervoso/metabolismo , Precursores de RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Síndromes de Usher/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células/genética , Corpos Enovelados/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/metabolismo , Proteômica , Precursores de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Fatores de Transcrição/metabolismo , Síndromes de Usher/genéticaRESUMO
The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare-/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.
Assuntos
Cílios/genética , Distrofias de Cones e Bastonetes/genética , Proteínas do Olho/genética , Segmento Externo da Célula Bastonete/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Animais , Cílios/patologia , Distrofias de Cones e Bastonetes/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Segmento Externo da Célula Bastonete/patologiaRESUMO
Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf-blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology.
Assuntos
Fatores de Processamento de RNA , Spliceossomos , Síndromes de Usher , Humanos , Proteínas do Olho/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Precursores de RNA/genética , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Spliceossomos/metabolismo , Fatores de Transcrição/metabolismo , Células HEK293RESUMO
The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer's disease.
Assuntos
Proteômica , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismo , Transdução de SinaisRESUMO
Mutations of the photoreceptor disc component (PRCD) gene are associated with rod-cone degeneration in both dogs and humans. Prcd is expressed in the mouse eye as early as embryonic day 14. In the adult mouse retina, PRCD is expressed in the outer segments of both rod and cone photoreceptors. Immunoelectron microscopy revealed that PRCD is located at the outer segment rim and that it is highly concentrated at the base of the outer segment. Prcd-knockout mice present with progressive retinal degeneration, starting at 20 weeks of age and onwards. This process is reflected by a significant and progressive reduction of both scotopic and photopic electroretinographic responses and by thinning of the retina, and specifically of the outer nuclear layer, indicating photoreceptor loss. Electron microscopy revealed severe damage to photoreceptor outer segments, which is associated with immigration of microglia cells to the Prcd-knockout retina and accumulation of vesicles in the inter-photoreceptor space. Phagocytosis of photoreceptor outer segment discs by the retinal pigmented epithelium is severely reduced. Our data show that Prcd-knockout mice serve as a good model for retinal degeneration caused by PRCD mutations in humans. Our findings in these mice support the involvement of PRCD in outer segment disc formation of both rod and cone photoreceptors. Furthermore, they suggest a feedback mechanism which coordinates the rate of photoreceptor outer segment disc formation, shedding and phagocytosis. This study has important implications for understanding the function of PRCD in the retina, as well as for future development of treatment modalities for PRCD deficiency in humans.
Assuntos
Distrofias de Cones e Bastonetes/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Segmento Externo da Célula Bastonete/patologia , Animais , Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/patologia , Proteínas do Olho/genética , Feminino , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Segmento Externo da Célula Bastonete/metabolismo , Transdução de SinaisRESUMO
The Usher syndrome (USH) is the most common form of inherited deaf-blindness, accompanied by vestibular dysfunction. Due to the heterogeneous manifestation of the clinical symptoms, three USH types (USH1-3) and additional atypical forms are distinguished. USH1 and USH2 proteins have been shown to function together in multiprotein networks in photoreceptor cells and hair cells. Mutations in USH proteins are considered to disrupt distinct USH protein networks and finally lead to the development of USH.To get novel insights into the molecular pathomechanisms underlying USH, we further characterize the periciliary USH protein network in photoreceptor cells. We show the direct interaction between the scaffold protein SANS (USH1G) and the transmembrane adhesion protein ush2a and that both assemble into a ternary USH1/USH2 complex together with the PDZ-domain protein whirlin (USH2D) via mutual interactions. Immunohistochemistry and proximity ligation assays demonstrate co-localization of complex partners and complex formation, respectively, in the periciliary region, the inner segment and at the synapses of rodent and human photoreceptor cells. Protein-protein interaction assays and co-expression of complex partners reveal that pathogenic mutations in USH1G severely affect formation of the SANS/ush2a/whirlin complex. Translational read-through drug treatment, targeting the c.728C > A (p.S243X) nonsense mutation, restored SANS scaffold function. We conclude that USH1 and USH2 proteins function together in higher order protein complexes. The maintenance of USH1/USH2 protein complexes depends on multiple USH1/USH2 protein interactions, which are disrupted by pathogenic mutations in USH1G protein SANS.
Assuntos
Surdocegueira/genética , Proteínas da Matriz Extracelular/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Síndromes de Usher/genética , Surdocegueira/patologia , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Humanos , Proteínas de Membrana/química , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Ligação Proteica , Mapas de Interação de Proteínas/genética , Estrutura Terciária de Proteína , Síndromes de Usher/complicações , Síndromes de Usher/patologiaRESUMO
The tegument of human cytomegalovirus (HCMV) virions contains proteins that interfere with both the intrinsic and the innate immunity. One protein with a thus far unknown function is pUL25. The deletion of pUL25 in a viral mutant (Towne-ΔUL25) had no impact on the release of virions and subviral dense bodies or on virion morphogenesis. Proteomic analyses showed few alterations in the overall protein composition of extracellular particles. A surprising result, however, was the almost complete absence of pUL26 in virions and dense bodies of Towne-ΔUL25 and a reduction of the large isoform pUL26-p27 in mutant virus-infected cells. pUL26 had been shown to inhibit protein conjugation with the interferon-stimulated gene 15 protein (ISG15), thereby supporting HCMV replication. To test for a functional relationship between pUL25 and pUL26, we addressed the steady-state levels of pUL26 and found them to be reduced in Towne-ΔUL25-infected cells. Coimmunoprecipitation experiments proved an interaction between pUL25 and pUL26. Surprisingly, the overall protein ISGylation was enhanced in Towne-ΔUL25-infected cells, thus mimicking the phenotype of a pUL26-deleted HCMV mutant. The functional relevance of this was confirmed by showing that the replication of Towne-ΔUL25 was more sensitive to beta interferon. The increase of protein ISGylation was also seen in cells infected with a mutant lacking the tegument protein pp65. Upon retesting, we found that pUL26 degradation was also increased when pp65 was unavailable. Our experiments show that both pUL25 and pp65 regulate pUL26 degradation and the pUL26-dependent reduction of ISGylation and add pUL25 as another HCMV tegument protein that interferes with the intrinsic immunity of the host cell.IMPORTANCE Human cytomegalovirus (HCMV) expresses a number of tegument proteins that interfere with the intrinsic and the innate defense mechanisms of the cell. Initial induction of the interferon-stimulated gene 15 protein (ISG15) and conjugation of proteins with ISG15 (ISGylation) by HCMV infection are subsequently attenuated by the expression of the viral IE1, pUL50, and pUL26 proteins. This study adds pUL25 as another factor that contributes to suppression of ISGylation. The tegument protein interacts with pUL26 and prevents its degradation by the proteasome. By doing this, it supports its restrictive influence on ISGylation. In addition, a lack of pUL25 enhances the levels of free ISG15, indicating that the tegument protein may interfere with the interferon response on levels other than interacting with pUL26. Knowledge obtained in this study widens our understanding of HCMV immune evasion and may also provide a new avenue for the use of pUL25-negative strains for vaccine production.
Assuntos
Citomegalovirus/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Células Cultivadas , Citocinas/metabolismo , Citomegalovirus/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Humanos , Imunidade Inata , Mutação , Fosfoproteínas/metabolismo , Proteólise , Proteômica/métodos , Ubiquitinas/metabolismo , Proteínas da Matriz Viral/metabolismo , Replicação ViralRESUMO
Despite the first successful applications of nonviral delivery vectors for small interfering RNA in the treatment of illnesses, such as the respiratory syncytial virus infection, the preparation of a clinically suitable, safe, and efficient delivery system still remains a challenge. In this study, we tackle the drawbacks of the existing systems by a combined experimental-computational in-depth investigation of the influence of the polymer architecture over the binding and transfection efficiency. For that purpose, a library of diblock copolymers with a molar mass of 30 kDa and a narrow dispersity (D < 1.12) was synthesized. We studied in detail the impact of an altered block size and/or composition of cationic diblock copolymers on the viability of each respective structure as a delivery agent for polynucleotides. The experimental investigation was further complemented by a computational study employing molecular simulations as well as an analytical description of systemic properties. This is the first report in which molecular dynamics simulations of RNA/cationic polymer complexes have been performed. Specifically, we developed and employed a coarse-grained model of the system at the molecular level to study the interactions between polymer chains and small interfering RNA. We were further able to confirm a threshold lengthbinding block/lengthnonbinding block ratio, which is required for efficient complexation of siRNA, and it was possible to find a correlation between the length of the cationic block and the size of the resulting polyplex. Hence, the combined insights from the experiments and the theoretical investigation resulted in a wealth of information about the properties of cationic diblock copolymers employed as RNA delivery agents, in particular regarding the molecular and mechanistic details of the interaction between the two components of a polyplex.
Assuntos
Simulação por Computador , Sistemas de Liberação de Medicamentos , Modelos Químicos , RNA Interferente Pequeno , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/farmacologiaRESUMO
Peripherin-2 is a glycomembrane protein exclusively expressed in the light-sensing compartments of rod and cone photoreceptors designated as outer segments (OS). Mutations in peripherin-2 are associated with degenerative retinal diseases either affecting rod or cone photoreceptors. While peripherin-2 has been extensively studied in rods, there is only little information on its supramolecular organization and function in cones. Recently, we have demonstrated that peripherin-2 interacts with the light detector rhodopsin in OS of rods. It remains unclear, however, if peripherin-2 also binds to cone opsins. Here, using a combination of co-immunoprecipitation analyses, transmission electron microscopy (TEM)-based immunolabeling experiments, and quantitative fluorescence resonance energy transfer (FRET) measurements in cone OS of wild type mice, we demonstrate that peripherin-2 binds to both, S-opsin and M-opsin. However, FRET-based quantification of the respective interactions indicated significantly less stringent binding of peripherin-2 to S-opsin compared to its interaction with M-opsin. Subsequent TEM-studies also showed less co-localization of peripherin-2 and S-opsin in cone OS compared to peripherin-2 and M-opsin. Furthermore, quantitative FRET analysis in acutely isolated cone OS revealed that the cone degeneration-causing V268I mutation in peripherin-2 selectively reduced binding to M-opsin without affecting the peripherin-2 interaction to S-opsin or rhodopsin. The differential binding of peripherin-2 to cone opsins and the mutant-specific interference with the peripherin-2/M-opsin binding points to a novel role of peripherin-2 in cones and might contribute to understanding the differential penetrance of certain peripherin-2 mutations in rods and cones. Finally, our results provide a proof-of-principle for quantitative FRET measurements of protein-protein interactions in cone OS.
Assuntos
Antígenos de Neoplasias/metabolismo , Opsinas dos Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Animais , Antígenos de Neoplasias/genética , Opsinas dos Cones/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Ligação Proteica , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/patologia , Rodopsina/genética , Rodopsina/metabolismoRESUMO
Usher syndrome is a genetically and clinically heterogeneous disease in humans, characterized by sensorineural hearing loss, retinitis pigmentosa and vestibular dysfunction. This disease is caused by mutations in genes encoding proteins that form complex networks in different cellular compartments. Currently, it remains unclear whether the Usher proteins also form networks within the olfactory epithelium (OE). Here, we describe Usher gene expression at the mRNA and protein level in the OE of mice and showed interactions between these proteins and olfactory signaling proteins. Additionally, we analyzed the odor sensitivity of different Usher syndrome mouse models using electro-olfactogram recordings and monitored significant changes in the odor detection capabilities in mice expressing mutant Usher proteins. Furthermore, we observed changes in the expression of signaling proteins that might compensate for the Usher protein deficiency. In summary, this study provides novel insights into the presence and purpose of the Usher proteins in olfactory signal transduction.
Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Odorantes/análise , Mucosa Olfatória/metabolismo , Olfato/genética , Síndromes de Usher/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Células Epiteliais/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Mucosa Olfatória/patologia , Transdução de Sinais , Síndromes de Usher/metabolismo , Síndromes de Usher/patologiaRESUMO
Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.
Assuntos
Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Degeneração Retiniana/genética , Síndromes de Usher/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra , Animais , Apoptose , Eletrorretinografia , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes , Técnicas de Genotipagem , Proteínas de Membrana/metabolismo , Microscopia Imunoeletrônica , Mutação , Retina/fisiopatologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Receptor do Retrovírus Politrópico e Xenotrópico , Proteínas de Peixe-Zebra/metabolismoRESUMO
Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cß-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant. The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Membrana/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Transporte Proteico/genética , Canais de Potencial de Receptor Transitório/genética , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Escuridão , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Olho/metabolismo , Luz , Proteínas de Membrana/metabolismo , Mutação , Rodopsina/metabolismo , Transdução de Sinais , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.
Assuntos
Moléculas de Adesão Celular/metabolismo , AMP Cíclico/fisiologia , Modelos Moleculares , Receptores Acoplados a Proteínas G/metabolismo , Sistemas do Segundo Mensageiro , Animais , Adesão Celular , Moléculas de Adesão Celular/química , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Movimento Celular , Humanos , Agências Internacionais , Ligantes , Farmacologia/tendências , Farmacologia Clínica/tendências , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/química , Isoformas de Proteínas/classificação , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/classificação , Transdução de Sinais , Sociedades Científicas , Terminologia como AssuntoRESUMO
The human Usher syndrome (USH) is a complex, rare disease manifesting in its most common form of inherited deaf-blindness. Due to the heterogeneous manifestation of the clinical symptoms, three clinical types (USH1-3) are distinguished according to the severity of the disease pattern. For a correct diagnosis, in addition to the auditory tests in early newborn screening, ophthalmological examinations and molecular genetic analysis are important. Ten known USH genes encode proteins, which are from heterogeneous protein families, interact in functional protein networks. In the eye and in the ear, USH proteins are expressed primarily in the mechano-sensitive hair cells and the rod and cone photoreceptor cells, respectively. In the hair cells, the USH protein networks are essential for the correct differentiation of the hair bundles as well as for the function of the mechano-electrical transduction complex in the matured cell. In the photoreceptor cells, USH proteins are located in the ciliary region and participate in intracellular transport processes. In addition, a USH protein network is present in the so-called calyceal processes. The lack of calyceal processes and the absence of a prominent visual phenotype in the mouse disqualifies mice as models for studies on the ophthalmic component of USH. While hearing impairments can be compensated with hearing aids and cochlear implants, there is no practical therapy for USH in the eye. Currently, gene-based therapy concepts, such as gene addition, applications of antisense oligonucleotides and TRIDs ("translational readthrough inducing drugs") for the readthrough of nonsense mutations are preclinically evaluated. For USH1B/MYO7A the UshStat gene therapy clinical trial is ongoing.
Assuntos
Ciliopatias/diagnóstico , Doenças Raras , Síndromes de Usher/diagnóstico , Animais , Ciliopatias/classificação , Ciliopatias/genética , Ciliopatias/terapia , Análise Mutacional de DNA , Surdocegueira/classificação , Surdocegueira/diagnóstico , Surdocegueira/genética , Surdocegueira/terapia , Modelos Animais de Doenças , Feminino , Humanos , Recém-Nascido , Camundongos , Triagem Neonatal , Células Fotorreceptoras de Vertebrados/fisiologia , Gravidez , Síndromes de Usher/classificação , Síndromes de Usher/genética , Síndromes de Usher/terapiaRESUMO
Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study, we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519C>T (p.R120X) into induced pluripotent stem cells (iPSC), and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells, suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization, Golgi cohesion and Gß1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren), we were able to restore up to 20% of endogenous, full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Células-Tronco Pluripotentes Induzidas/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Mutação , Biossíntese de Proteínas , Epitélio Pigmentado da Retina/citologia , Diferenciação Celular , Reprogramação Celular , Cílios/metabolismo , Cílios/patologia , Proteínas do Olho/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Oxidiazóis/farmacologia , Fenótipo , Biossíntese de Proteínas/efeitos dos fármacos , Transporte Proteico , Adulto JovemRESUMO
In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with "cone-first" retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs(∗)2];[Glu529Valfs(∗)2], and c.[401delT(;)3354G>A], p.[Leu134Argfs(∗)45(;)Trp1118(∗)]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543(∗)) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families.
Assuntos
Proteínas de Transporte/genética , Peptídeo Sintases/genética , Distrofias Retinianas/genética , Adulto , Alelos , Animais , Feminino , Genes Recessivos , Variação Genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , LinhagemRESUMO
Deafblindness is part of several genetic disorders. We investigated a consanguineous Egyptian family with two siblings affected by congenital hearing loss and retinal degeneration, initially diagnosed as Usher syndrome type 1. At teenage, severe enamel dysplasia, developmental delay, and microcephaly became apparent. Genome-wide homozygosity mapping and whole-exome sequencing detected a homozygous missense mutation, c.1238G>T (p.Gly413Val), affecting a highly conserved residue of peroxisomal biogenesis factor 6, PEX6. Biochemical profiling of the siblings revealed abnormal and borderline plasma phytanic acid concentration, and cerebral imaging revealed white matter disease in both. We show that Pex6 localizes to the apical extensions of secretory ameloblasts and differentiated odontoblasts at early stages of dentin synthesis in mice, and to cilia of retinal photoreceptor cells. We propose PEX6, and possibly other peroxisomal genes, as candidate for the rare cooccurrence of deafblindness and enamel dysplasia. Our study for the first time links peroxisome biogenesis disorders to retinal ciliopathies.
Assuntos
Adenosina Trifosfatases/genética , Surdocegueira/genética , Hipoplasia do Esmalte Dentário/genética , Microcefalia/genética , Mutação de Sentido Incorreto , Degeneração Retiniana/genética , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/metabolismo , Ameloblastos/metabolismo , Ameloblastos/patologia , Sequência de Aminoácidos , Animais , Criança , Cílios/metabolismo , Cílios/patologia , Consanguinidade , Surdocegueira/metabolismo , Surdocegueira/patologia , Hipoplasia do Esmalte Dentário/metabolismo , Hipoplasia do Esmalte Dentário/patologia , Feminino , Expressão Gênica , Homozigoto , Humanos , Masculino , Camundongos , Microcefalia/metabolismo , Microcefalia/patologia , Dados de Sequência Molecular , Odontoblastos/metabolismo , Odontoblastos/patologia , Linhagem , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Irmãos , Substância Branca/metabolismo , Substância Branca/patologia , Adulto JovemRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Síndromes de Usher/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Clatrina/genética , Clatrina/metabolismo , Endocitose , Regulação da Expressão Gênica , Guanilato Quinases , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Fosforilação , Células Fotorreceptoras de Vertebrados/patologia , Cultura Primária de Células , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Síndromes de Usher/metabolismo , Síndromes de Usher/patologiaRESUMO
Mutations in the FAM161A gene were previously identified as the cause for autosomal-recessive retinitis pigmentosa 28. To study the effects of Fam161a dysfunction in vivo, we generated gene-trapped Fam161a(GT/GT) mice with a disruption of its C-terminal domain essential for protein-protein interactions. We confirmed the absence of the full-length Fam161a protein in the retina of Fam161a(GT/GT) mice using western blots and showed weak expression of a truncated Fam161a protein by immunohistochemistry. Histological analyses demonstrated that photoreceptor segments were disorganized in young Fam161a(GT/GT) mice and that the outer retina was completely lost at 6 months of age. Reactive microglia appeared in the outer retina and electroretinography showed an early loss of photoreceptor function in 4-month-old Fam161a(GT/GT) animals. Light and electron microscopy revealed a remarkable phenotype of a significantly shortened connecting cilium, spread ciliary microtubule doublets and disturbed disk organization in Fam161a(GT/GT) photoreceptor cells. Co-immunolabeling experiments demonstrated reduced expression and mislocalization of centrin 3 and disturbed targeting of the Fam161a interactors lebercilin and Cep290, which were restricted to the basal body and proximal connecting cilium in Fam161a(GT/GT) retinas. Moreover, we identified misrouting of the outer segment cargo proteins opsin and rds/peripherin 2 in Fam161a(GT/GT) mice. In conclusion, our results suggest a critical role for the C-terminal domain of Fam161a for molecular interactions and integrity of the connecting cilium. Fam161a is required for the molecular delivery into the outer segment cilium, a function which is essential for outer segment disk formation and ultimately visual function.