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2.
Hum Mol Genet ; 26(24): 4896-4905, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29036441

RESUMO

Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of dominant retinitis pigmentosa (RP), a type of inherited blindness caused by the dysfunction and death of photoreceptor cells. The P23H mutation, the most frequent single cause of RP in the USA, causes rhodopsin misfolding and induction of the unfolded protein response (UPR), an adaptive ER stress response and signalling network that aims to enhance the folding and degradation of misfolded proteins to restore proteostasis. Prolonged UPR activation, and in particular the PERK branch, can reduce protein synthesis and initiate cell death through induction of pro-apoptotic pathways. Here, we investigated the effect of pharmacological PERK inhibition on retinal disease process in the P23H-1 transgenic rat model of retinal degeneration. PERK inhibition with GSK2606414A led to an inhibition of eIF2α phosphorylation, which correlated with reduced ERG function and decreased photoreceptor survival at both high and low doses of PERK inhibitor. Additionally, PERK inhibition increased the incidence of inclusion formation in cultured cells overexpressing P23H rod opsin, and increased rhodopsin aggregation in the P23H-1 rat retina, suggesting enhanced P23H misfolding and aggregation. In contrast, treatment of P23H-1 rats with an inhibitor of eIF2α phosphatase, salubrinal, led to improved photoreceptor survival. Collectively, these data suggest the activation of PERK is part of a protective response to mutant rhodopsin that ultimately limits photoreceptor cell death.


Assuntos
Retinose Pigmentar/metabolismo , Rodopsinas Sensoriais/metabolismo , eIF-2 Quinase/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Humanos , Indóis/farmacologia , Dobramento de Proteína , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/genética , Rodopsinas Sensoriais/genética , Estresse Fisiológico/fisiologia , Resposta a Proteínas não Dobradas , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética
3.
Hum Mol Genet ; 26(2): 305-319, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28065882

RESUMO

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Metformina/administração & dosagem , Degeneração Retiniana/genética , Retinose Pigmentar/genética , Rodopsina/genética , Proteínas Quinases Ativadas por AMP/biossíntese , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Proteínas Mutantes/genética , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/patologia , Dobramento de Proteína/efeitos dos fármacos , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologia , Ratos , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/tratamento farmacológico , Retinose Pigmentar/patologia , Rodopsina/química , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Segmento Externo da Célula Bastonete/patologia , Ativação Transcricional/efeitos dos fármacos
4.
Cell Stem Cell ; 18(6): 769-781, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27151457

RESUMO

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention.


Assuntos
Cegueira/patologia , Cegueira/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Padrões de Herança/genética , Disco Óptico/citologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Cílios/efeitos dos fármacos , Cílios/metabolismo , Proteínas do Citoesqueleto , Éxons/genética , Proteínas do Olho/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Amaurose Congênita de Leber/patologia , Masculino , Morfolinos/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Opsinas/metabolismo , Organogênese/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/ultraestrutura , Proteínas rab de Ligação ao GTP/metabolismo
5.
Hum Mol Genet ; 24(4): 972-86, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25292197

RESUMO

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 Jovem
6.
Hum Mutat ; 35(11): 1354-62, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25168334

RESUMO

Mutations in the OPN1LW (L-) and OPN1MW (M-)cone opsin genes underlie a spectrum of cone photoreceptor defects from stationary loss of color vision to progressive retinal degeneration. Genotypes of 22 families with a range of cone disorders were grouped into three classes: deletions of the locus control region (LCR); missense mutation (p.Cys203Arg) in an L-/M-hybrid gene; and exon 3 single-nucleotide polymorphism (SNP) interchange haplotypes in an otherwise normal gene array. Moderate-to-high myopia was observed in all mutation categories. Individuals with LCR deletions or p.Cys203Arg mutations were more likely to have nystagmus and poor vision, with disease progression in some p.Cys203Arg patients. Three disease-associated exon 3 SNP haplotypes encoding LIAVA, LVAVA, or MIAVA were identified in our cohort. These patients were less likely to have nystagmus but more likely to show progression, with all patients over the age of 40 years having marked macular abnormalities. Previously, the haplotype LIAVA has been shown to result in exon 3 skipping. Here, we show that haplotypes LVAVA and MIAVA also result in aberrant splicing, with a residual low level of correctly spliced cone opsin. The OPN1LW/OPN1MW:c.532A>G SNP, common to all three disease-associated haplotypes, appears to be principally responsible for this mutational mechanism.


Assuntos
Opsinas dos Cones/genética , Estudos de Associação Genética , Genótipo , Mutação , Fenótipo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Criança , Pré-Escolar , Ordem dos Genes , Inativação Gênica , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Haplótipos , Hemizigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Oftalmoscópios , Linhagem , Polimorfismo de Nucleotídeo Único , Splicing de RNA , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/genética , Deleção de Sequência , Adulto Jovem
7.
Hum Mol Genet ; 23(24): 6594-606, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25055872

RESUMO

Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Many rod opsin mutations, such as P23H, lead to misfolding of rod opsin with detrimental effects on photoreceptor function and viability. Misfolded P23H rod opsin and other mutations in the intradiscal domain are characterized by the formation of an incorrect disulphide bond between C185 and C187, as opposed to the correct and highly conserved C110-C187 disulphide bond. Therefore, we tested the hypothesis that incorrect disulphide bond formation might be a factor that affects the biogenesis of rod opsin by studying wild-type (WT) or P23H rod opsin in combination with amino acid substitutions that prevent the formation of incorrect disulphide bonds involving C185. These mutants had altered traffic dynamics, suggesting a requirement for regulation of disulphide bond formation/reduction during rod opsin biogenesis. Here, we show that the BiP co-chaperone and reductase protein ERdj5 (DNAJC10) regulates this process. ERdj5 overexpression promoted the degradation, improved the endoplasmic reticulum mobility and prevented the aggregation of P23H rod opsin. ERdj5 reduction by shRNA delayed rod opsin degradation and promoted aggregation. The reductase and co-chaperone activity of ERdj5 were both required for these effects on P23H rod opsin. Furthermore, mutations in these functional domains acted as dominant negatives that affected WT rod opsin biogenesis. Collectively, these data identify ERdj5 as a member of the proteostasis network that regulates rod opsin biogenesis and supports a role for disulphide bond formation/reduction in rod opsin biogenesis and disease.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Neurônios/metabolismo , Rodopsina/genética , Linhagem Celular Tumoral , Dissulfetos/química , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP40/antagonistas & inibidores , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/metabolismo , Mutação , Neurônios/citologia , Plasmídeos/química , Plasmídeos/metabolismo , Agregados Proteicos , Dobramento de Proteína , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopsina/metabolismo , Transdução de Sinais , Transfecção
8.
Hum Mol Genet ; 23(8): 2164-75, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24301679

RESUMO

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(-/-) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.


Assuntos
Predisposição Genética para Doença , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Mutação/genética , Piridonas/farmacologia , Pirimidinas/farmacologia , Retinose Pigmentar/prevenção & controle , Rodopsina/metabolismo , Animais , Western Blotting , Células Cultivadas , Eletrorretinografia , Feminino , Receptor Quinase 1 Acoplada a Proteína G/genética , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Genes Dominantes , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina/genética , Tomografia de Coerência Óptica , Visão Ocular/efeitos dos fármacos , Visão Ocular/fisiologia
9.
PLoS One ; 8(8): e73944, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24023695

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1(G93A) transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1(G93A) and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1(G93A) aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Fármacos Neuroprotetores/metabolismo , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Peso Corporal , Bovinos , Sobrevivência Celular , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Longevidade , Masculino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neurônios Motores/patologia , Músculos/fisiopatologia , Tamanho do Órgão , Estrutura Quaternária de Proteína , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Ubiquitinação
10.
Mol Biol Cell ; 23(18): 3522-31, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22855534

RESUMO

Mutations in rod opsin-the light-sensitive protein of rod cells-cause retinitis pigmentosa. Many rod opsin mutations lead to protein misfolding, and therefore it is important to understand the role of molecular chaperones in rod opsin biogenesis. We show that BiP (HSPA5) prevents the aggregation of rod opsin. Cleavage of BiP with the subtilase cytotoxin SubAB results in endoplasmic reticulum (ER) retention and ubiquitylation of wild-type (WT) rod opsin (WT-green fluorescent protein [GFP]) at the ER. Fluorescence recovery after photobleaching reveals that WT-GFP is usually mobile in the ER. By contrast, depletion of BiP activity by treatment with SubAB or coexpression of a BiP ATPase mutant, BiP(T37G), decreases WT-GFP mobility to below that of the misfolding P23H mutant of rod opsin (P23H-GFP), which is retained in the ER and can form cytoplasmic ubiquitylated inclusions. SubAB treatment of P23H-GFP-expressing cells decreases the mobility of the mutant protein further and leads to ubiquitylation throughout the ER. Of interest, BiP overexpression increases the mobility of P23H-GFP, suggesting that it can reduce mutant rod opsin aggregation. Therefore inhibition of BiP function results in aggregation of rod opsin in the ER, which suggests that BiP is important for maintaining the solubility of rod opsin in the ER.


Assuntos
Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Opsinas de Bastonetes/metabolismo , Western Blotting , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacologia , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Microscopia Confocal , Mutação , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Opsinas de Bastonetes/genética , Subtilisinas/metabolismo , Subtilisinas/farmacologia , Transfecção , Ubiquitinação/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
11.
Hum Mol Genet ; 21(16): 3647-54, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22619378

RESUMO

X-linked retinitis pigmentosa (XLRP) is genetically heterogeneous with two causative genes identified, RPGR and RP2. We previously mapped a locus for a severe form of XLRP, RP23, to a 10.71 Mb interval on Xp22.31-22.13 containing 62 genes. Candidate gene screening failed to identify a causative mutation, so we adopted targeted genomic next-generation sequencing of the disease interval to determine the molecular cause of RP23. No coding variants or variants within or near splice sites were identified. In contrast, a variant deep within intron 9 of OFD1 increased the splice site prediction score 4 bp upstream of the variant. Mutations in OFD1 cause the syndromic ciliopathies orofaciodigital syndrome-1, which is male lethal, Simpson-Golabi-Behmel syndrome type 2 and Joubert syndrome. We tested the effect of the IVS9+706A>G variant on OFD1 splicing in vivo. In RP23 patient-derived RNA, we detected an OFD1 transcript with the insertion of a cryptic exon spliced between exons 9 and 10 causing a frameshift, p.N313fs.X330. Correctly spliced OFD1 was also detected in patient-derived RNA, although at reduced levels (39%), hence the mutation is not male lethal. Our data suggest that photoreceptors are uniquely susceptible to reduced expression of OFD1 and that an alternative disease mechanism can cause XLRP. This disease mechanism of reduced expression for a syndromic ciliopathy gene causing isolated retinal degeneration is reminiscent of CEP290 intronic mutations that cause Leber congenital amaurosis, and we speculate that reduced dosage of correctly spliced ciliopathy genes may be a common disease mechanism in retinal degenerations.


Assuntos
Mutação da Fase de Leitura , Proteínas/genética , Retinose Pigmentar/etiologia , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos X , Éxons , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Sítios de Splice de RNA , Retinose Pigmentar/genética , Análise de Sequência de DNA
12.
PLoS One ; 7(2): e30866, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22347407

RESUMO

Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Olho/fisiologia , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Humanos , Amaurose Congênita de Leber , Mutação , Retina/patologia , Fatores de Transcrição
14.
Am J Hum Genet ; 87(1): 26-39, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20579627

RESUMO

X-linked cone and cone-rod dystrophies (XLCOD and XLCORD) are a heterogeneous group of progressive disorders that solely or primarily affect cone photoreceptors. Mutations in exon ORF15 of the RPGR gene are the most common underlying cause. In a previous study, we excluded RPGR exon ORF15 in some families with XLCOD. Here, we report genetic mapping of XLCOD to Xq26.1-qter. A significant LOD score was detected with marker DXS8045 (Z(max) = 2.41 [theta = 0.0]). The disease locus encompasses the cone opsin gene array on Xq28. Analysis of the array revealed a missense mutation (c. 529T>C [p. W177R]) in exon 3 of both the long-wavelength-sensitive (LW, red) and medium-wavelength-sensitive (MW, green) cone opsin genes that segregated with disease. Both exon 3 sequences were identical and were derived from the MW gene as a result of gene conversion. The amino acid W177 is highly conserved in visual and nonvisual opsins across species. We show that W177R in MW opsin and the equivalent W161R mutation in rod opsin result in protein misfolding and retention in the endoplasmic reticulum. We also demonstrate that W177R misfolding, unlike the P23H mutation in rod opsin that causes retinitis pigmentosa, is not rescued by treatment with the pharmacological chaperone 9-cis-retinal. Mutations in the LW/MW cone opsin gene array can, therefore, lead to a spectrum of disease, ranging from color blindness to progressive cone dystrophy (XLCOD5).


Assuntos
Opsinas dos Cones/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Células Fotorreceptoras Retinianas Cones/patologia , Doenças Retinianas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Cromossomos Humanos X/genética , Feminino , Estudos de Associação Genética , Ligação Genética , Loci Gênicos , Haplótipos , Humanos , Escore Lod , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Estrutura Secundária de Proteína , Doenças Retinianas/patologia , Doenças Retinianas/fisiopatologia
15.
Hum Mol Genet ; 19(12): 2421-32, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20332100

RESUMO

Nance-Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell-cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development.


Assuntos
Actinas/metabolismo , Adesões Focais/metabolismo , Proteínas Nucleares/metabolismo , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Sequência de Aminoácidos , Animais , Células CACO-2 , Citoesqueleto/metabolismo , Adesões Focais/ultraestrutura , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas Nucleares/genética , Estrutura Terciária de Proteína/genética , Pseudópodes/ultraestrutura , Ratos , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética
16.
J Cell Sci ; 122(Pt 24): 4465-72, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19934218

RESUMO

Mutations in rod opsin, the archetypal G-protein-coupled receptor, cause retinitis pigmentosa. The majority of mutations, e.g. P23H, cause protein misfolding, resulting in ER retention, induction of the unfolded protein response and degradation by ERAD. If misfolded rod opsin escapes degradation, it aggregates and forms intracellular inclusions. Therefore, it is important to identify the chaperones that mediate the folding or degradation of rod opsin. ER degradation enhancing alpha-mannosidase-like 1 (EDEM1) can enhance the release of terminally misfolded glycoproteins from the calnexin chaperone system. Here, we identify EDEM1 as a novel chaperone of rod opsin. EDEM1 expression promoted the degradation of P23H rod opsin and decreased its aggregation. By contrast, shRNA-mediated knockdown of EDEM1 increased both the amount of P23H rod opsin and its aggregation into inclusions. EDEM1 was detected in rod photoreceptor inner segments and EndoH-sensitive rod opsin co-immunoprecipitated with EDEM1 from retina, suggesting that rod opsin is a physiological EDEM1 client. Unexpectedly, EDEM1 binding to rod opsin was independent of mannose trimming and EDEM1 promoted the cell-surface expression of mutant rod opsin. Collectively, the data suggest that EDEM1 is a chaperone for rod opsin and that expression of EDEM1 can be used to promote correct folding, as well as enhanced degradation, of mutant proteins in the ER to combat protein-misfolding disease.


Assuntos
Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Rodopsina/metabolismo , Humanos , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Transporte Proteico , Retina/metabolismo , Rodopsina/química , Rodopsina/genética
17.
Mol Vis ; 15: 876-84, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19421413

RESUMO

PURPOSE: To perform a phenotypic assessment of members of three British families with blue cone monochromatism (BCM), and to determine the underlying molecular genetic basis of disease. METHODS: Affected members of three British families with BCM were examined clinically and underwent detailed electrophysiological and psychophysical testing. Blood samples were taken for DNA extraction. Molecular analysis involved the amplification of the coding regions of the long (L) and medium (M) wave cone opsin genes and the upstream locus control region (LCR) by polymerase chain reaction (PCR). Gene products were directly sequenced and analyzed. RESULTS: In all three families, genetic analysis identified that the underlying cause of BCM involved an unequal crossover within the opsin gene array, with an inactivating mutation. Family 1 had a single 5'-L-M-3' hybrid gene, with an inactivating Cys203Arg (C203R) mutation. Family 3 had an array composed of a C203R inactivated 5'-L-M-3' hybrid gene followed by a second inactive gene. Families 1 and 3 had typical clinical, electrophysiological, and psychophysical findings consistent with stationary BCM. A novel mutation was detected in Family 2 that had a single hybrid gene lacking exon 2. This family presented clinical and psychophysical evidence of a slowly progressive phenotype. CONCLUSIONS: Two of the BCM-causing family genotypes identified in this study comprised different hybrid genes, each of which contained the commonly described C203R inactivating mutation. The genotype in the family with evidence of a slowly progressive phenotype represents a novel BCM mutation. The deleted exon 2 in this family is not predicted to result in a shift in the reading frame, therefore we hypothesize that an abnormal opsin protein product may accumulate and lead to cone cell loss over time. This is the first report of slow progression associated with this class of mutation in the L or M opsin genes in BCM.


Assuntos
Defeitos da Visão Cromática/genética , Opsinas/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Eletrorretinografia , Família , Feminino , Deleção de Genes , Inativação Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Reino Unido
18.
Hum Mol Genet ; 18(14): 2643-55, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19414485

RESUMO

Nance-Horan syndrome (NHS) is an X-linked developmental disorder characterized by congenital cataract, dental anomalies, facial dysmorphism and, in some cases, mental retardation. Protein truncation mutations in a novel gene (NHS) have been identified in patients with this syndrome. We previously mapped X-linked congenital cataract (CXN) in one family to an interval on chromosome Xp22.13 which encompasses the NHS locus; however, no mutations were identified in the NHS gene. In this study, we show that NHS and X-linked cataract are allelic diseases. Two CXN families, which were negative for mutations in the NHS gene, were further analysed using array comparative genomic hybridization. CXN was found to be caused by novel copy number variations: a complex duplication-triplication re-arrangement and an intragenic deletion, predicted to result in altered transcriptional regulation of the NHS gene. Furthermore, we also describe the clinical and molecular analysis of seven families diagnosed with NHS, identifying four novel protein truncation mutations and a novel large deletion encompassing the majority of the NHS gene, all leading to no functional protein. We therefore show that different mechanisms, aberrant transcription of the NHS gene or no functional NHS protein, lead to different diseases. Our data highlight the importance of copy number variation and non-recurrent re-arrangements leading to different severity of disease and describe the potential mechanisms involved.


Assuntos
Catarata/genética , Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Proteínas Nucleares/genética , Adulto , Sequência de Bases , Catarata/congênito , Catarata/metabolismo , Criança , Pré-Escolar , Feminino , Dosagem de Genes , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Humanos , Recém-Nascido , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Linhagem , Adulto Jovem
19.
Invest Ophthalmol Vis Sci ; 45(8): 2786-94, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15277505

RESUMO

PURPOSE: To use porcine lens capsule (PLC) as basement membrane for ARPE-19 cells and to characterize its effects on cell differentiation and gene expression. METHODS: Postconfluent cultures of ARPE-19 cells were established on either porous polyester filters or PLC membranes and characterized by electron microscopy, immunocytochemistry, and transepithelial electrical resistance measurements. Metabolic activity was assessed by measuring phagocytosis of rod outer segments. mRNA populations of ARPE-19 cells grown on polyester and PLC membranes were compared by suppressive subtractive hybridization. Differentially regulated messages were subsequently identified by DNA sequencing and their altered expression confirmed by Northern or virtual Northern blot analysis. RESULTS: Culture of ARPE-19 cells on PLC membrane induced the formation of apical microvilli and the ability to phagocytose rod outer segments. These culture conditions also led to enhanced junctional distribution of ZO-1 and occludin, the formation of polarized membrane domains, and a significant increase in transepithelial resistance. Gene expression was significantly altered by growth on PLC membranes and 29 differentially expressed transcripts were identified. CONCLUSIONS: Culture of ARPE-19 cells on PLC membranes resulted in a more differentiated phenotype and in expression of a specific set of transcripts encoding protein products that may affect epithelial differentiation, polarity and survival.


Assuntos
Membrana Basal/fisiologia , Proteínas do Olho/genética , Regulação da Expressão Gênica/fisiologia , Cápsula do Cristalino/fisiologia , Epitélio Pigmentado Ocular/citologia , Animais , Northern Blotting , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Polaridade Celular/fisiologia , Sobrevivência Celular/fisiologia , Condutividade Elétrica , Proteínas do Olho/metabolismo , Biblioteca Gênica , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Ocludina , Fagocitose/fisiologia , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Epitélio Pigmentado Ocular/metabolismo , RNA Mensageiro/metabolismo , Segmento Externo da Célula Bastonete/fisiologia , Suínos , Proteína da Zônula de Oclusão-1
20.
Invest Ophthalmol Vis Sci ; 45(2): 675-84, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14744914

RESUMO

PURPOSE: Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration. The cell line ARPE-19 was therefore examined for response to oxidative stress and its effect on stress protein induction and junctional integrity. METHODS: ARPE-19 cell viability after 1 week or 5 weeks in culture was assessed in response to different concentrations of hydrogen peroxide. The response to sublethal doses was assessed by examination of heme oxygenase (HO)-1, Hsp27 and Hsp70 by immunofluorescence and Western blot analysis. Immunofluorescence was used to investigate the localization of the junctional proteins zonula occludens (ZO)-1, occludin, and N-cadherin, and beta-catenin. Subcellular fractionation was used to assess any redistribution of beta-catenin. Monolayer integrity was examined by measurement of flux of rhodamine-conjugated dextrans from the apical to basal aspect of cells. RESULTS: ARPE-19 cells cultured for 5 weeks were less sensitive to chronic oxidative stress induced by hydrogen peroxide than those cultured for 1 week. The more differentiated ARPE-19 cells had higher steady state levels of Hsp27 and Hsp70. The response to stress also differed with time in culture. The localization of junctional proteins, which became strongly peripheral after 5 weeks in culture, became disrupted after oxidative stress, and cytosolic beta-catenin increased. Chronic oxidative stress also increased paracellular flux across the monolayer. CONCLUSIONS: Increased resistance to chronic oxidative stress with differentiation in ARPE-19 cells correlated with higher steady state levels of Hsp27 and Hsp70. Oxidative stress disrupted RPE cell junction and barrier integrity, which may contribute to the pathogenesis of diseases related to RPE through disruption of the blood-retinal barrier.


Assuntos
Proteínas de Choque Térmico , Estresse Oxidativo , Epitélio Pigmentado Ocular/patologia , Junções Íntimas/patologia , Barreira Hematorretiniana/efeitos dos fármacos , Western Blotting , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Humanos , Peróxido de Hidrogênio/toxicidade , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Ocludina , Oxidantes/toxicidade , Fosfoproteínas/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/metabolismo , Transativadores/metabolismo , Proteína da Zônula de Oclusão-1 , beta Catenina
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