Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Front Neurol ; 14: 1207616, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37448751

RESUMO

Background: The balance between the activity of the Na+/K+/Cl- cotransporter (NKCC1) that introduces Cl- into the cell and the K+/Cl- cotransporter (KCC2) that transports Cl- outside the cell is critical in determining the inhibitory or excitatory outcome of GABA release. Mounting evidence suggests that the impairment of GABAergic inhibitory neurotransmission plays a crucial role in the pathophysiology of epilepsy, both in patients and animal models. Previous studies indicate that decreased KCC2 expression is linked to audiogenic seizures in GASH/Sal hamsters, highlighting that Cl- imbalance can cause neuronal hyperexcitability. In this study, we aimed to investigate whether the Na+/K+/Cl- cotransporter NKCC1 is also affected by audiogenic seizures and could, therefore, play a role in neuronal hyperexcitability within the GASH/Sal epilepsy model. Methods: NKCC1 protein expression in both the GASH/Sal strain and wild type hamsters was analyzed by immunohistochemistry and Western blotting techniques. Brain regions examined included cortex, hippocampus, hypothalamus, inferior colliculus and pons-medulla oblongata, which were evaluated both at rest and after sound-inducing seizures in GASH/Sal hamsters. A complementary analysis of NKCC1 gene slc12a2 expression was conducted by real-time PCR. Finally, protein and mRNA levels of glutamate decarboxylase GAD67 were measured as an indicator of GABA release. Results: The induction of seizures caused significant changes in NKCC1 expression in epileptic GASH/Sal hamsters, despite the similar brain expression pattern of NKCC1 in GASH/Sal and wild type hamsters in the absence of seizures. Interestingly, the regulation of brain NKCC1 by seizures demonstrated regional specificity, as protein levels exclusively increased in the hippocampus and hypothalamus. Complementary real-time PCR analysis revealed that NKCC1 regulation was post-transcriptional only in the hypothalamus. In addition, seizures also modulated GAD67 mRNA levels in a brain region-specific manner. The increased GAD67 expression in the hippocampus and hypothalamus of the epileptic hamster brain suggests that NKCC1 upregulation overlaps with GABA release in these regions during seizures. Conclusions: Our results indicate that seizure induction causes dysregulation of NKCC1 expression in GASH/Sal animals, which overlaps with changes in GABA release. These observations provide evidence for the critical role of NKCC1 in how seizures affect neuronal excitability, and support NKCC1 contribution to the development of secondary foci of epileptogenic activity.

2.
Int J Mol Sci ; 23(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36077382

RESUMO

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.


Assuntos
Anormalidades do Olho , Peixe-Zebra , Animais , Proteínas do Citoesqueleto , Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hipertrofia , Masculino , Camundongos , Camundongos Transgênicos , Retina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
3.
HLA ; 99(3): 160-166, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34997833

RESUMO

The characterization of the expression profile of HLA questionable alleles (Q) is clinically relevant in allogeneic hematopoietic stem cell transplantation (HSTC) because an aberrant expression of these alleles could lead to transplantation-related complications. HLA-DQB1*03:01:01:21Q shows a substitution at the donor splice site of intron 3 that potentially could affect the expression of this allele. In order to determine their expression profile at RNA and protein level, we analyzed the presence of the HLA-DQ7 molecule by complement-dependent cytotoxicity test (CDC) and flow cytometry, and their RNA processing by cDNA analyses and sequencing by Sanger methods. Our results reveal that HLA-DQ7 is not detectable by serological methods, this is confirmed by cDNA methods demonstrating the absence of specific HLA-DQB1*03:01:01:21Q mRNA, probably due to an intron 3 retention that creates a premature TGA stop codon, leading to mRNA degradation via nonsense-mediated decay (NMD). These findings demonstrate that the HLA-DQB1*03:01:01:21Q allele is nonexpressed, thus it has been renamed as DQB1*03:01:01:21N.


Assuntos
RNA , Alelos , DNA Complementar/genética , Cadeias beta de HLA-DQ/genética , Humanos
4.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208498

RESUMO

CYP1B1 loss of function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle; however, the underlying molecular mechanisms are poorly understood. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in the 72% mRNA reduction with the residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Microphthalmia and jaw maldevelopment were observed in 23% of F0 somatic mosaic mutant larvae (144 hpf). These early phenotypes were not detected in cyp1b1-KO F3 larvae (144 hpf), but 27% of adult (four months) zebrafish exhibited uni- or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in cyp1b1 mutants. Transcriptomic analyses of the offspring (seven dpf) of cyp1b1-KO progenitors with adult-onset craniofacial defects revealed functionally enriched differentially expressed genes related to extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids and fatty acids and oxidation-reduction processes that include several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of the phenotypes and molecular pathways associated with cyp1b1 LoF, with species dependency, and provides evidence for the dysregulation of extracellular matrix gene expression as one of the mechanisms underlying the pathogenicity associated with cyp1b1 disruption.


Assuntos
Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Matriz Extracelular/genética , Estudos de Associação Genética , Metabolismo dos Lipídeos/genética , Animais , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Camundongos Transgênicos , Peixe-Zebra
5.
Biology (Basel) ; 10(2)2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573230

RESUMO

Myocilin is a secreted glycoprotein with a poorly understood biological function and it is mainly known as the first glaucoma gene. To explore the normal role of this protein in vivo we developed a myoc knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries a homozygous variant (c.236_239delinsAAAGGGGAAGGGGA) that is predicted to result in a loss-of-function of the protein because of a premature termination codon p.(V75EfsX60) that resulted in a significant reduction of myoc mRNA levels. Immunohistochemistry showed the presence of myocilin in wild-type embryonic (96 h post-fertilization) anterior segment eye structures and caudal muscles. The protein was also detected in different adult ocular and non-ocular tissues. No gross macroscopic or microscopic alterations were identified in the KO zebrafish, but, remarkably, we observed absence of females among the adult KO animals and apoptosis in the immature juvenile gonad (28 dpf) of these animals, which is characteristic of male development. Transcriptomic analysis showed that adult KO males overexpressed key genes involved in male sex determination and presented differentially expressed Wnt signalling genes. These results show that myocilin is required for ovary differentiation in zebrafish and provides in vivo support for the role of myocilin as a Wnt signalling pathway modulator. In summary, this myoc KO zebrafish line can be useful to investigate the elusive function of this protein, and it provides evidence for the unexpected function of myocilin as a key factor in zebrafish sex determination.

6.
Genes (Basel) ; 11(5)2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32422965

RESUMO

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.


Assuntos
Glaucoma/genética , Proteínas Ativadoras de Guanilato Ciclase/fisiologia , Retina/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Adulto , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Glaucoma/congênito , Gliose/genética , Gliose/patologia , Proteínas Ativadoras de Guanilato Ciclase/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
7.
Hum Genet ; 139(10): 1209-1231, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32274568

RESUMO

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.


Assuntos
Complemento C3/genética , Matriz Extracelular/metabolismo , Anormalidades do Olho/genética , Glaucoma/genética , Mutação com Perda de Função , Inibidor da Tripsina Pancreática de Kazal/genética , alfa-Macroglobulinas/genética , Adulto , Animais , Câmara Anterior/metabolismo , Câmara Anterior/patologia , Câmara Anterior/cirurgia , Sistemas CRISPR-Cas , Estudos de Casos e Controles , Complemento C3/deficiência , Embrião não Mamífero , Matriz Extracelular/patologia , Anormalidades do Olho/metabolismo , Anormalidades do Olho/patologia , Anormalidades do Olho/cirurgia , Feminino , Edição de Genes , Expressão Gênica , Genes Recessivos , Glaucoma/metabolismo , Glaucoma/patologia , Glaucoma/cirurgia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Malha Trabecular/cirurgia , Trabeculectomia , Inibidor da Tripsina Pancreática de Kazal/deficiência , Peixe-Zebra , alfa-Macroglobulinas/deficiência
8.
PLoS One ; 14(1): e0211029, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30657791

RESUMO

Congenital glaucoma (CG) is a severe and inherited childhood optical neuropathy that leads to irreversible visual loss and blindness in children. CG pathogenesis remains largely unexplained in most patients. Herein we have extended our previous studies to evaluate the role of FOXC2 and PITX2 variants in CG. Variants of the proximal promoter and transcribed sequence of these two genes were analyzed by Sanger sequencing in a cohort of 133 CG families. To investigate possible oligogenic inheritance involving FOXC2 or PITX2 and CYP1B1, we also analyzed FOXC2 and PITX2 variants in a group of 25 CG cases who were known to carry CYP1B1 glaucoma-associated genotypes. The functional effect of three identified variants was assessed by transactivation luciferase reporter assays, protein stability and subcellular localization analyses. We found eight probands (6.0%) who carried four rare FOXC2 variants in the heterozygous state. In addition, we found an elevated frequency (8%) of heterozygous and rare PITX2 variants in the group of CG cases who were known to carry CYP1B1 glaucoma-associated genotypes, and one of these PITX2 variants arose de novo. To the best of our knowledge, two of the identified variants (FOXC2: c.1183C>A, p.(H395N); and PITX2: c.535C>A, p.(P179T)) have not been previously identified. Examination of the genotype-phenotype correlation in this group suggests that the presence of the infrequent PITX2 variants increase the severity of the phenotype. Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: p.(C498R) and p.(H395N); PITX2: p.(P179T)). In summary, the increased frequency in PCG patients of rare FOXC2 and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG.


Assuntos
Fatores de Transcrição Forkhead , Glaucoma , Proteínas de Homeodomínio , Herança Multifatorial , Mutação de Sentido Incorreto , Fatores de Transcrição , Substituição de Aminoácidos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Glaucoma/congênito , Glaucoma/metabolismo , Células HEK293 , Heterozigoto , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Homeobox PITX2
9.
PLoS One ; 13(12): e0209364, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30557320

RESUMO

Myocilin is an extracellular glycoprotein with a poorly understood biological function and typically known because of its association with glaucoma. In this study, we analyzed the expression and biological activity of human myocilin in some non-ocular tissues. Western immunoblot showed the presence of myocilin in blood plasma as well as in liver and lymphoid tissues (thymus and lymph node). Quantitative PCR confirmed the expression of MYOC in these lymphoid organs and revealed that its mRNA is also present in T-lymphocytes and leukocytes. In addition, detection of 30 kDa C-terminal myocilin fragments in thymus and liver suggested that myocilin undergoes an in vivo proteolytic processing that might regulate its biological activity. The presence of myocilin in blood was further corroborated by peptide mass fingerprinting of the HPLC-isolated protein, and gross estimation of its concentration by Western immunoblot indicated that it is a medium-abundance serum protein with an approximate concentration of 0.85 mg/ml (15.5 µM). Finally, in vitro analyses indicated that myocilin acts as an anti-adhesive protein for human circulating leukocytes incubated with endothelial cell monolayers. Altogether, these data provide insightful information on new biological properties of myocilin and suggest its putative role as a blood matricellular protein.


Assuntos
Proteínas Sanguíneas/fisiologia , Adesão Celular , Proteínas do Citoesqueleto/fisiologia , Proteínas do Olho/fisiologia , Glicoproteínas/fisiologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Leucócitos/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Sanguíneas/análise , Western Blotting , Proteínas do Citoesqueleto/sangue , Proteínas do Olho/sangue , Feminino , Glicoproteínas/sangue , Células HEK293 , Voluntários Saudáveis , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Proteólise , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Timo/metabolismo
10.
Sci Rep ; 7: 46175, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28397860

RESUMO

Congenital glaucoma (CG) is a heterogeneous, inherited and severe optical neuropathy that originates from maldevelopment of the anterior segment of the eye. To identify new disease genes, we performed whole-exome sequencing of 26 unrelated CG patients. In one patient we identified two rare, recessive and hypermorphic coding variants in GPATCH3, a gene of unidentified function, and 5% of a second group of 170 unrelated CG patients carried rare variants in this gene. The recombinant GPATCH3 protein activated in vitro the proximal promoter of CXCR4, a gene involved in embryo neural crest cell migration. The GPATCH3 protein was detected in human tissues relevant to glaucoma (e.g., ciliary body). This gene was expressed in the dermis, skeletal muscles, periocular mesenchymal-like cells and corneal endothelium of early zebrafish embryos. Morpholino-mediated knockdown and transient overexpression of gpatch3 led to varying degrees of goniodysgenesis and ocular and craniofacial abnormalities, recapitulating some of the features of zebrafish embryos deficient in the glaucoma-related genes pitx2 and foxc1. In conclusion, our data suggest the existence of high genetic heterogeneity in CG and provide evidence for the role of GPATCH3 in this disease. We also show that GPATCH3 is a new gene involved in ocular and craniofacial development.


Assuntos
Proteínas de Transporte/genética , Sequenciamento do Exoma , Olho/embriologia , Face/embriologia , Glaucoma/congênito , Glaucoma/genética , Mutação/genética , Crânio/embriologia , Animais , Segregação de Cromossomos/genética , Embrião não Mamífero/metabolismo , Família , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Linhagem , Fenótipo , Regiões Promotoras Genéticas/genética , Receptores CXCR4/genética , Frações Subcelulares/metabolismo , Ativação Transcricional/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética
11.
PLoS One ; 12(4): e0176386, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28448622

RESUMO

Mutations in the CYP1B1 gene are currently the main known genetic cause of primary congenital glaucoma (PCG), a leading cause of blindness in children. Here, we analyze for the first time the CYP1B1 genotype activity and the microscopic and clinical phenotypes in human PCG. Surgical pieces from trabeculectomy from patients with PCG (n = 5) and sclerocorneal rims (n = 3) from cadaver donors were processed for transmission electron microscopy. Patients were classified into three groups depending on goniodysgenesis severity, which was influenced by CYP1B1 enzymatic activity. The main histological changes observed in the outflow pathway of patients with PCG and mutations in CYP1B1 were: i) underdeveloped collector channels and the Schlemm's canal; ii) abnormal insertion of the ciliary muscle; iii) death of the trabecular endothelial cells. Our findings could be useful in improving treatment strategy of PCG associated with CYP1B1 mutations.


Assuntos
Citocromo P-450 CYP1B1/genética , Genótipo , Glaucoma/congênito , Glaucoma/genética , Adolescente , Criança , Feminino , Glaucoma/complicações , Glaucoma/enzimologia , Humanos , Masculino , Mutação , Linhagem , Fenótipo
12.
Acta Ophthalmol ; 94(7): e555-e560, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27060699

RESUMO

PURPOSE: To evaluate the function of eight missense CYP1B1 single nucleotide variants (SNVs) previously identified in patients with primary congenital glaucoma (PCG). METHODS: The eight variants were obtained by site-directed mutagenesis and transiently expressed in human embryonic kidney 293-T (HEK-293T) cells. The catalytic activity, protein stability and subcellular localization of the different recombinant CYP1B1 variants were assessed in this cell line. RESULTS: Six of the mutant CYP1B1 proteins (p.L89P, p.A106D, p.R390S, p.P437L, p.C470Y and S485F) showed catalytic activity values ranging from 0% to 4% of those of the wild-type protein and were considered null variants. The activity values of the two remaining variants (p.F123L and p.A237E) were close to 20% of that of the wild-type enzyme and were classified as hypomorphic variants. Reduced protein stability contributed partially to the decreased catalytic activity of two of the mutant enzymes (p.L89P and p.A106D). None of the CYP1B1 variants showed intracellular aggregation and they all displayed a normal subcellular localization in the endoplasmic reticulum, suggesting that they had folded into a wild-type-like structure. The enzymatic activity associated with the different genotypes in which these CYP1B1 variants were present was estimated to range from 0% to 10% of that of the wild-type genotype. CONCLUSION: These results confirm the pathogenicity of the analysed missense CYP1B1 variants and further support the concept that either absent or very low CYP1B1 activity levels are the primary molecular defect involved in PCG pathogenesis.


Assuntos
Citocromo P-450 CYP1B1/genética , Hidroftalmia/genética , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único , Western Blotting , Citocromo P-450 CYP1B1/metabolismo , Primers do DNA/química , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Regulação Enzimológica da Expressão Gênica/fisiologia , Genótipo , Células HEK293 , Humanos , Lactente , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Transfecção
13.
Eur J Hum Genet ; 24(5): 672-80, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26220699

RESUMO

Primary congenital glaucoma (PCG) is the cause of a significant proportion of inherited visual loss in children, but the underlying mechanism is poorly understood. In this study, we assessed the relationship between PCG and FOXC1 variants by Sanger sequencing the proximal promoter and transcribed sequence of FOXC1 from a cohort of 133 PCG families with no known CYP1B1 or MYOC mutations. The pathogenicity of the identified variants was evaluated by functional analyses. Ten patients (7.5%) with no family history of glaucoma carried five different rare heterozygous FOXC1 variants with both increased (rs77888940:C>G, c.-429C>G, rs730882054:c.1134_144del(CGGCGGCGCGG), p.(G380Rfs*144) and rs35717904:A>T, c.*734A>T) and decreased (rs185790394: C>T, c.-244C>T and rs79691946:C>T, p.(P297S)) transactivation, ranging from 50 to 180% of the wild-type activity. The five variants did not show monogenic segregation, and four of them were absent in a control group (n=233). To the best of our knowledge, one of these variants (p.(G380Rfs*144)) has not previously been described. One of the FOXC1 variant carriers (p.(P297S)) also coinherited a functionally altered rare PITX2 heterozygous variant (rs6533526:C>T, c.*454C>T). Bioinformatics and functional analyses provided novel information on three of these variants. c.-429C>G potentially disrupts a consensus sequence for a terminal oligopyrimidine tract, whereas c.-244C>T may alter the RNA secondary structure in the 5'-untranslated region (UTR) that affects mRNA translation. In addition, p.(G380Rfs*144) led to increased protein stability. In summary, these data reveal the presence of translation regulatory sequences in the UTRs of FOXC1 and provide evidence for a possible role of rare FOXC1 variants as modifying factors of goniodysgenesis in PCG.


Assuntos
Regiões 5' não Traduzidas , Fatores de Transcrição Forkhead/genética , Glaucoma/congênito , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Pré-Escolar , Feminino , Fatores de Transcrição Forkhead/metabolismo , Glaucoma/genética , Células HEK293 , Heterozigoto , Proteínas de Homeodomínio/genética , Humanos , Lactente , Masculino , Estabilidade Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Proteína Homeobox PITX2
14.
Microrna ; 4(1): 50-6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25809640

RESUMO

Mutations of the FOXC1 transcription factor are involved in a variety of autosomal dominant ocular anterior segment defects, ranging from Axenfeld-Rieger malformations to isolated glaucoma in some patients. In this study we have evaluated the possible role of the c.*734A>T FOXC1 variant as a modifier factor of the activity of two FOXC1 mutations previously identified in families primarily affected by dominant glaucoma (haplotypes p.G447_G448insDG-c.*734A>T and p.I126S-c.*734A>T). Previous bioinformatic analyses indicated that the c.*734A>T variant is located in a potential target sequence for hsa-miR-548l. Co-expression of this miRNA with a reporter cDNA construct in which the wild-type 3'UTR sequence of FOXC1 was fused to the 3'-end of the firefly luciferase coding region, led to approximately 20% decreased luciferase activity compared to the controls, confirming the presence of a target sequence for hsa-miR-548l. In contrast, this miRNA did not show any effect on the luciferase activity associated with the mutant 3'UTR FOXC1 sequence, showing that it resulted in a loss-of-function of the has-miR-548l target sequence. In addition, functional evaluation of the two glaucoma-associated haplotypes revealed increased protein levels and transactivation, compared to the corresponding individual coding mutations (approximately 1.2-fold on average). These data support the role of hsa-miR-548l as a regulator of FOXC1 translation and provide evidence for the c.*734A>T variant as a modifier factor for the activity of coding glaucoma-associated FOXC1 mutations.


Assuntos
Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Glaucoma/genética , MicroRNAs/genética , Mutação , Biossíntese de Proteínas , Sequência de Bases , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética
15.
PLoS One ; 10(3): e0119272, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25786029

RESUMO

Dominant glaucoma, a heterogeneous, infrequent and irreversible optic neuropathy, is often associated with elevated intraocular pressure and early-onset. The role of FOXC1 in this type of glaucoma was investigated in twelve Spanish probands via nucleotide variation screening of its proximal promoter and unique exon. Functional evaluations of the identified variants included analyses of the transcriptional activity, protein stability, DNA binding ability and subcellular localization. Four different mutations that were identified in four probands (33.3%) were associated with remarkable phenotypic variability and were functionally classified as either hypermorphic (p.Y47X, p.Q106X and p.G447_G448insDG) or hypomorphic (p.I126S) alleles. To the best of our knowledge, three of the variants are novel (p.Y47X, p.I126S and p.G447_G448insDG) and, in addition, hypermorphic FOXC1 mutations are reported herein for the first time. The presence of an intact N-terminal activation domain in the truncated proteins p.Y47X and p.Q106X may underlie their associated transactivation hyperactivity by a gain-of-function mechanism involving dysregulated protein-protein interactions. Similarly, altered molecular interactions may also lead to increased p.G447_G448insDG activity. In contrast, the partial loss-of-function associated with p.I126S was due to impaired protein stability, DNA binding, protein phosphorylation and subcellular distribution. These results support that moderate and variable FOXC1 transactivation changes are associated with moderate goniodysgenesis, dominant glaucoma and remarkable phenotypic variability.


Assuntos
Fatores de Transcrição Forkhead/genética , Glaucoma/genética , Mutação , Fenótipo , Ativação Transcricional , Adulto , Idoso , Sequência de Aminoácidos , Criança , Pré-Escolar , Feminino , Glaucoma/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Alinhamento de Sequência , Análise de Sequência de DNA
16.
PLoS One ; 8(1): e54385, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23342144

RESUMO

Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc.) affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.


Assuntos
Bicarbonatos/farmacologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Humor Aquoso/metabolismo , Western Blotting , Linhagem Celular , Meios de Cultura/farmacologia , Humanos , Proteólise/efeitos dos fármacos
17.
Invest Ophthalmol Vis Sci ; 52(1): 179-89, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20926826

RESUMO

PURPOSE: Myocilin is an extracellular glycoprotein with unknown function that is associated with glaucoma. Calpain II cleaves recombinant myocilin within the linker region of the protein, releasing the C-terminal olfactomedin domain from the N-terminal domain. The authors previously reported that myocilin interacts with the C-terminal region of hevin, a secretory glycoprotein belonging to the SPARC family of matricellular proteins. This study aims to investigate the interaction of myocilin with SPARC. METHODS: Protein-protein interactions were evaluated by the yeast two-hybrid system. The positive interactions were confirmed by solid-phase binding assays using Ni-chelating HPLC purified recombinant proteins and coexpression of recombinant proteins in HEK-293T cells. Coexpression of myocilin, SPARC, and hevin in ocular tissues was identified by immunoflorescence microscopy, Western blot, and array-based gene profiling. RESULTS: Yeast two-hybrid analyses showed that myocilin interacted with the highly conserved C-terminal extracellular calcium binding (EC) domain within SPARC and hevin. Solid-phase binding assays confirmed these interactions and showed that both myocilin and its C-terminal olfactomedin fragment interacted noncovalently with SPARC and a peptide containing the EC domain of SPARC. Full-length myocilin interacted with higher affinity with SPARC and its EC domain than the myocilin C-terminal fragment. Coexpression of the two recombinant proteins in HEK-293T cells also indicated their intracellular interaction. CONCLUSIONS: Recombinant myocilin and SPARC interact through their C-terminal domains. The data suggest that the proteolytic processing of myocilin modulates this interaction as well as the interactions of myocilin with other extracellular matrix and matricellular proteins, further supporting a functional role for this proteolytic cleavage.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Osteonectina/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Western Blotting , Proteínas de Ligação ao Cálcio/química , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Proteínas do Citoesqueleto/química , Proteínas da Matriz Extracelular/química , Proteínas do Olho/química , Perfilação da Expressão Gênica , Glicoproteínas/química , Humanos , Rim/embriologia , Microscopia de Fluorescência , Dados de Sequência Molecular , Osteonectina/química , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Técnicas do Sistema de Duplo-Híbrido
18.
Invest Ophthalmol Vis Sci ; 51(1): 72-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19696176

RESUMO

PURPOSE: Recombinant myocilin expressed in cells in culture is endoproteolytically cleaved in the endoplasmic reticulum by calpain II, releasing an N-terminal and a C-terminal fragment. This proteolytic processing has been speculated to regulate the molecular interactions of myocilin. The main purpose of this study was to analyze the effect of the proteolytic cleavage on myocilin aggregation. METHODS: cDNAs encoding human myocilin and the N- and C-terminal fragments were transiently expressed in HEK-293T cells. Covalent interactions of recombinant myocilin were analyzed by SDS-PAGE and Western immunoblot analysis in different dissociating conditions. Noncovalent interactions were studied by solid-phase binding assays, performed with Ni-chelating HPLC-purified recombinant proteins, and by Far-Western blot analysis. RESULTS: Western blot analysis of recombinant myocilin aggregates under either increasing ionic strength or increasing concentration of reducing agent indicated that ionic interactions do not contribute to the stability of the molecular complexes linked by disulfide bridges. Disulfide myocilin homoaggregates decreased as the proteolytic processing increased. Solid-phase binding assays showed the existence of high-affinity (K(d) = 0.068 microM) noncovalent myocilin-myocilin interactions and that processed fragments bound to the full-length protein with significantly reduced affinity. Far-Western blot analysis confirmed noncovalent interactions between recombinant myocilin disulfide aggregates. CONCLUSIONS: The proteolytic processing of recombinant myocilin decreases myocilin homoaggregates. These data provide the first evidence of a functional role for this processing in myocilin aggregation and suggest that disulfide complexes of myocilin could organize into a dynamic extracellular network sustained by noncovalent N-terminal interactions.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Olho/química , Glicoproteínas/química , Proteínas Recombinantes/química , Animais , Segmento Anterior do Olho/metabolismo , Western Blotting , Bovinos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Rim/embriologia , Concentração Osmolar , Ligação Proteica , Esclera/metabolismo , Transfecção
19.
Mol Vis ; 14: 2097-108, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19023451

RESUMO

PURPOSE: Heterozygous mutations in the myocilin gene (MYOC) cause glaucoma by an unknown mechanism. MYOC encodes an extracellular protein of unidentified function that undergoes intracellular endoproteolytic processing in the secretory pathway. It has been described that co-expression of wild-type/mutant myocilin reduces the secretion of the wild-type protein and that single expression of glaucoma myocilin mutants reduces its proteolytic processing. However, the effect of wild-type myocilin on mutant myocilin secretion and how mutant myocilin affects the proteolytic processing of wild-type myocilin have not been investigated. We herein analyze these two issues. METHODS: We modeled the heterozygous state for 4 missense (E323K, R346T, P370L, D380A) and 1 nonsense (Q368X) myocilin mutants by transiently co-expressing each mutant with the wild-type protein in HEK-293T cells. Recombinant mutant and wild-type myocilin in both culture media and cellular fractions were quantified by western immunoblot and densitometry. RESULTS: A 24 h transient co-expression of each myocilin mutant with the wild-type protein elicited an augmented secretion of the mutant forms from 1.5 fold (D380A) to 5.4 fold (E323K). Under such conditions, extracellular mutant myocilin represented up to 20% of the total mutant protein. Other than this effect, secreted wild-type myocilin significantly decreased from 2.6 fold (E323K) to 36 fold (Q368X). When myocilin proteolytic processing was enhanced (96 hour co-expression) the extracellular amount of wild-type processed myocilin diminished from approximately 2.1 fold (E323K) to 6.3 fold (P370L). Nonreducing SDS-PAGE indicated that extracellular myocilin resulting from 24 h co-expression of wild-type myocilin and each of the 4 missense mutants forms hetero-oligomers and that glaucoma mutations do not increase the size of myocilin aggregates. CONCLUSIONS: Increased extracellular levels of mutant myocilin expressed in heterozygosis may play a relevant role in glaucoma pathogenesis. This effect is likely the result of intracellular mutant/wild-type myocilin hetero-oligomerization.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Espaço Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glaucoma/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Heterozigoto , Proteínas Mutantes/metabolismo , Processamento de Proteína Pós-Traducional , Linhagem Celular , Proteínas do Citoesqueleto/química , DNA Complementar/genética , Proteínas do Olho/química , Glicoproteínas/química , Humanos , Modelos Biológicos , Proteínas Mutantes/genética , Mutação/genética , Estrutura Quaternária de Proteína
20.
J Biotechnol ; 134(1-2): 193-201, 2008 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-18282627

RESUMO

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.


Assuntos
Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serpinas/metabolismo , Animais , Western Blotting , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas do Olho/genética , Humanos , Microscopia de Fluorescência , Fatores de Crescimento Neural/genética , Pichia/genética , Serpinas/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...