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1.
PLoS One ; 15(1): e0227859, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935276

RESUMO

In order to provide a cost-effective method to narrow down the number of pathogenic Crystallin beta A4 (CRYBA4) non-synonymous single nucleotide polymorphisms (nsSNPs), we collected nsSNP information of the CRYBA4 gene from SNP databases and literature, predicting the pathogenicity and possible changes of protein properties and structures using multiple bioinformatics tools. The nsSNP data of the CRYBA4 gene were collected from 4 databases and published literature. According to 12 criteria, six bioinformatics tools were chosen to predict the pathogenicity. I-Mutant 2.0, Mupro and INPS online tools were used to analyze the effects of amino acid substitution on protein stability by calculating the value of ΔΔG. ConSurf, SOPMA, GETAREA and HOPE online tools were used to predict the evolutionary conservation of amino acids, solvent accessible surface areas, and the physical and chemical properties and changes of protein structure. All 157 CRYBA4 nsSNPs were analyzed. Forty-four CRYBA4 high-risk pathogenic nsSNPs (predicted to be pathogenic by all six software tools) were detected out of the 157 CRYBA4 nsSNPs, four of which (c.283C>T, p.R95W; c.449T>A, p.V150D; c.475G>A, p.G159R; c.575G>C, p.R192P) should be focused on because of their high potential pathogenicity and possibility of changing protein properties. Thirty high-risk nsSNPs were predicted to cause a decrease of protein stability. Twenty-nine high-risk nsSNPs occurred in evolutionary conserved positions. Twenty-two high-risk nsSNPs occurred in the core of the protein. It is predicted that these high-risk pathogenic nsSNPs can cause changes in the physical and chemical properties of amino acids, resulting in structural changes of proteins and changes in the interactions between domains and other molecules, thus affecting the function of proteins. This study provides important reference value when narrowing down the number of pathogenic CRYBA4 nsSNPs and studying the pathogenesis of congenital cataracts. By using this method, we can easily find 44 high-risk pathogenic nsSNPs out of 157 CRYBA4 nsSNPs.


Assuntos
Catarata/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Cadeia A de beta-Cristalina/genética , Substituição de Aminoácidos/genética , Catarata/congênito , Catarata/patologia , Biologia Computacional , Simulação por Computador , Estudos de Associação Genética , Humanos , Mutação , Fenótipo , Conformação Proteica , Estabilidade Proteica , Software , Cadeia A de beta-Cristalina/química
2.
BMC Med Genet ; 20(1): 153, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488069

RESUMO

BACKGROUND: Mutations in more than 52 genes have been identified in isolated congenital cataracts, the majority of which are located in crystalline and connexin (gap junction) genes. An in-frame one amino acid deletion in the beta-crystalline gene CRYBA1 has been reported in several different Chinese, Caucasian and Iranian families of congenital cataracts. Further functional studies are needed to confirm the variant pathogenicity. METHODS: The purpose of this study is to identify the genetic causes that contribute to congenital cataracts with esotropia and nystagmus in a Chinese family. Whole-exome sequencing was performed on samples from all five family members. The two brothers of the father and their daughters were then enrolled in the study, and 40 suspected variants were sequenced among the 9 subjects using Sanger sequencing. The mRNA and protein levels of CRYBA1 in the lens epithelium from cataract patients and normal controls were compared using quantitative polymerase chain reaction (qPCR) and Western blot analyses. The wild-type and mutated forms (p.G91del) of CRYBA1 cDNA were transfected into two types of cell lines, and the expression level of exogenous CRYBA1 was measured by Western blot analysis. The exogenous CRYBA1 proteins were visualized by immunofluorescence staining. RESULTS: In this two-generation family, all three descendants inherited congenital cataracts with esotropia and nystagmus from the father, while the mother's lens was normal. After two rounds of sequencing, CRYBA1 (c. 269-271 del, p.G91del) was identified as the mutation responsible for the autosomal dominant congenital cataract in the Chinese family. CRYBA1 showed lower expression in cataract lenses than in control lenses. The deleted form (p.G91del) of CRYBA1 showed lower expression and was more aggregate to the cell membrane than the wild-type CRYBA1. CONCLUSIONS: We performed molecular experiments to confirm that the p.G91del mutation in CRYBA1 results in abnormal expression and distribution of CRYBA1 protein, and this study could serve as an example of the pathogenicity of an in-frame small deletion in an inherited eye disorder.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Catarata/congênito , Catarata/genética , Predisposição Genética para Doença/genética , Deleção de Sequência , Cadeia A de beta-Cristalina/genética , Idoso , Sequência de Bases , Catarata/diagnóstico por imagem , Catarata/patologia , Linhagem Celular , Grupo com Ancestrais do Continente Europeu , Feminino , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Sequenciamento Completo do Exoma , Cadeia A de beta-Cristalina/metabolismo
3.
Exp Eye Res ; 186: 107712, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31254514

RESUMO

Crystallins are structural proteins in the lens that last a lifetime with little turnover. Deviant in crystallins can cause rare but severe visual impairment, namely, congenital cataracts. It is reported that several mutations in the acidic ß-crystallin 4 (CRYBA4) are related to congenital cataracts. However, the pathogenesis of these mutants is not well understood at molecular level. Here we evaluate the biochemical properties of wild type CRYBA4 (CRYBA4WT) and a pathogenic G64W mutant (CRYBA4G64W) including protein folding, polymerization state and protein stability. Furthermore, we explore the differences in their interactions with α-crystallin A (CRYAA) and basic ß-crystallin 1 (CRYBB1) via yeast two-hybrid and pull-down assay in vitro, through which we find that G64W mutation leads to protein misfolding, decreases protein stability, blocks its interaction with CRYBB1 but maintains its interaction with CRYAA. Our results deepen our understanding of the pathogenesis of congenital cataracts.


Assuntos
Catarata , Cristalino/metabolismo , Dobramento de Proteína , Cadeia A de beta-Cristalina/genética , beta-Cristalinas/química , Catarata/congênito , Catarata/genética , Catarata/metabolismo , Humanos , Mutação
4.
Invest Ophthalmol Vis Sci ; 60(1): 234-244, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30646012

RESUMO

Purpose: Crystallin gene expression during lens fiber cell differentiation is tightly spatially and temporally regulated. A significant fraction of mammalian genes is transcribed from adjacent promoters in opposite directions ("bidirectional" promoters). It is not known whether two proximal genes located on the same allele are simultaneously transcribed. Methods: Mouse lens transcriptome was analyzed for paired genes whose transcriptional start sites are separated by less than 5 kbp to identify coexpressed bidirectional promoter gene pairs. To probe these transcriptional mechanisms, nascent transcription of Cryba4, Crybb1, and Crybb3 genes from gene-rich part of chromosome 5 was visualized by RNA fluorescent in situ hybridizations (RNA FISH) in individual lens fiber cell nuclei. Results: Genome-wide lens transcriptome analysis by RNA-seq revealed that the Cryba4-Crybb1 pair has the highest Pearson correlation coefficient between their steady-state mRNA levels. Analysis of Cryba4 and Crybb1 nascent transcription revealed frequent simultaneous expression of both genes from the same allele. Nascent Crybb3 transcript visualization in "early" but not "late" differentiating lens fibers show nuclear accumulation of the spliced Crybb3 transcripts that was not affected in abnormal lens fiber cell nuclei depleted of chromatin remodeling enzyme Snf2h (Smarca5). Conclusions: The current study shows for the first time that two highly expressed lens crystallin genes, Cryba4 and Crybb1, can be simultaneously transcribed from adjacent bidirectional promoters and do not show nuclear accumulation. In contrast, spliced Crybb3 mRNAs transiently accumulate in early lens fiber cell nuclei. The gene pairs coexpressed during lens development showed significant enrichment in human "cataract" phenotype.


Assuntos
Cristalinas/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Núcleo do Cristalino/embriologia , RNA Mensageiro/genética , Fatores de Transcrição/fisiologia , Cadeia A de beta-Cristalina/genética , Cadeia B de beta-Cristalina/genética , Animais , Diferenciação Celular , Feminino , Hibridização in Situ Fluorescente , Camundongos
5.
Gene ; 692: 113-118, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30659945

RESUMO

The transcription factor v-maf avain musculoaponeurotic fibrosarcoma oncogene homolog (MAF) plays an important role in lens development. It contains a unique extended homology region (EHR) in the DNA binding domain. MAF mutations are associated with phenotypically distinct forms of congenital cataract and show different effects on the transactivation of target genes. Mutations in the MAF EHR region were rarely reported and their corresponding phenotype and impact on target genes' transactivation were not evaluated. A three- generation Chinese family with congenital cataract was recruited. The patients in the family present non-syndromic congenital nuclear and lamellar opacities. A novel MAF mutation (c.812 T > A, p.Val271Glu) was identified by targeted next-generation sequencing. The mutation is in highly conserved EHR region of MAF and co-segregates with the cataract in the family. It is predicted to be pathogenic by multiple algorithms and is absent in a control population. Dual luciferase activity assay shows the mutation significantly impair the transcriptional activity of four crystallin genes (CRYAA, CRYBA4, CRYBA1, and CRYGA) and two non-crystallin genes (HMOX1 and KDELR2). Herein, we report a novel missense mutation in the MAF EHR region of the DNA binding domain in a family with congenital cataract. The mutation is associated with non-syndromic bilateral nuclear cataract and impacts the transactivation of cataract associated genes involved in lens structure and stress response.


Assuntos
Catarata/genética , Cristalinas/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-maf/genética , Sítios de Ligação , Catarata/patologia , Catarata/terapia , Extração de Catarata , Feminino , Heme Oxigenase-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Domínios Proteicos , Proteínas Proto-Oncogênicas c-maf/metabolismo , Ativação Transcricional , Proteínas de Transporte Vesicular/genética , Cadeia A de beta-Cristalina/genética
6.
Oral Dis ; 25(1): 274-281, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29683234

RESUMO

OBJECTIVE: Masticatory muscle tendon-aponeurosis hyperplasia, which is associated with limited mouth opening, progresses very slowly from adolescence. The prevalence rates of this disease are higher among women than among men, suggesting oestrogen involvement. As parafunctional habits are frequently observed, mechanical stress is likely involved in the pathogenesis and advancement of this disease. To elucidate the pathological condition, we examined the effect of oestrogen on tenocyte function and the relationship between mechanical stress and crystallin beta A4 (Cryba4), using murine TT-D6 tenocytes. MATERIALS AND METHODS: Cell proliferation assays, RT-PCR, real-time RT-PCR, Western blot analysis and mechanical loading experiments were performed. RESULTS: The physiological dose of oestrogen increased the levels of scleraxis and tenomodulin in TT-D6 tenocytes. In contrast, forced expression of Cryba4 inhibited scleraxis expression in these cells. Surprisingly, oestrogen significantly promoted cell differentiation in the Cryba4-overexpressing TT-D6 tenocytes. Moreover, tensile force induced Cryba4 expression in these tendon cells. CONCLUSION: Oestrogen and Cryba4 may be associated with the progression of masticatory muscle tendon-aponeurosis hyperplasia.


Assuntos
Aponeurose/patologia , Estrogênios/fisiologia , Músculos da Mastigação/patologia , Tendões/patologia , Cadeia A de beta-Cristalina/genética , Animais , Células Cultivadas , Humanos , Hiperplasia , Camundongos , Estresse Mecânico
7.
PLoS One ; 13(12): e0204968, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30543633

RESUMO

The alsodid ground frogs of the Eupsophus genus are divided into two groups, the roseus (2n = 30) and vertebralis (2n = 28), which are distributed throughout the temperate Nothofagus forests of South America. Currently, the roseus group is composed by four species, while the vertebralis group consists of two. Phylogenetic relationships and species delimitation within each group are controversial. In fact, previous analyses considered that the roseus group was composed of between four to nine species. In this work, we evaluated phylogenetic relationships, diversification times, and species delimitation within the roseus group using a multi-locus dataset. For this purpose, mitochondrial (D-loop, Cyt b, and COI) and nuclear (POMC and CRYBA1) partial sequences from 164 individuals were amplified, representing all species. Maximum Likelihood (ML) and Bayesian approaches were used to reconstruct phylogenetic relationships. Species tree was estimated using BEAST and singular value decomposition scores for species quartets (SVDquartets). Species limits were evaluated with six coalescent approaches. Diversification times were estimated using mitochondrial and nuclear rates with LogNormal relaxed clock in BEAST. Nine well-supported monophyletic lineages were recovered in Bayesian, ML, and SVDquartets, including eight named species and a lineage composed by specimens from the Villarrica population (Bootstrap:>70, PP:> 0.99). Single-locus species delimitation analyses overestimated the species number in E. migueli, E. calcaratus, and E. roseus lineages, while multi-locus analyses recovered as species the nine lineages observed in phylogenetic analyses (Ctax = 0.69). It is hypothesized that Eupsophus diversification occurred during Mid-Pleistocene (0.42-0.14 Mya), with most species having originated after the Last Southern Patagonian Glaciation (0.18 Mya). Our results revitalize the hypothesis that the E. roseus group is composed of eight species and support the Villarrica lineage as a new putative species.


Assuntos
Proteínas de Anfíbios/genética , Anuros/classificação , Anuros/genética , Filogenia , Pró-Opiomelanocortina/genética , Cadeia A de beta-Cristalina/genética , Animais , Chile , Especificidade da Espécie
8.
Invest Ophthalmol Vis Sci ; 59(4): AMD104-AMD113, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30098172

RESUMO

Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that ßA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells. Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of ßA3/A1-crystallin. Results: ßA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of ßA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. ßA3/A1-crystallin binds to cortactin, and loss of ßA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls. Conclusions: We introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that ßA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.


Assuntos
Cristalinas/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Atrofia Geográfica/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Cadeia A de beta-Cristalina/fisiologia , Animais , Western Blotting , Movimento Celular/fisiologia , Humanos , Imuno-Histoquímica , Camundongos Knockout , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição da Família Snail/genética , Transfecção , Vimentina/genética , Cicatrização/fisiologia
9.
Sci Rep ; 8(1): 5944, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29654292

RESUMO

For many neurodegenerative disorders, expression of a pathological protein by one cell type impedes function of other cell types, which in turn contributes to the death of the first cell type. In transgenic mice modelling Stargardt-like (STGD3) maculopathy, human mutant ELOVL4 expression by photoreceptors is associated with defects in the underlying retinal pigment epithelium (RPE). To examine how photoreceptors exert cytotoxic effects on RPE cells, transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates were studied one month prior (preclinical stage) to onset of photoreceptor loss (two months). TG photoreceptor outer segments presented to human RPE cells are recognized and internalized into phagosomes, but their digestion is delayed. Live RPE cell imaging pinpoints decreased numbers of acidified phagolysomes. In vivo, master regulator of lysosomal genes, transcription factor EB (TFEB), and key lysosomal enzyme Cathepsin D are both unaffected. Oxidative stress, as ruled out with high-resolution respirometry, does not play a role at such an early stage. Upregulation of CRYBA1/A3 and phagocytic cells (microglia/macrophages) interposed between RPE and photoreceptors support adaptive responses to processing delays. Impaired phagolysosomal maturation is observed in RPE of mice expressing human mutant ELOVL4 in their photoreceptors prior to photoreceptor death and associated vision loss.


Assuntos
Lisossomos/patologia , Degeneração Macular/congênito , Fagossomos/patologia , Células Fotorreceptoras/patologia , Epitélio Pigmentado da Retina/patologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Catepsina D/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Humanos , Lisossomos/metabolismo , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/fisiologia , Fagossomos/metabolismo , Células Fotorreceptoras/metabolismo , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Epitélio Pigmentado da Retina/metabolismo , Cadeia A de beta-Cristalina/metabolismo
10.
Curr Eye Res ; 43(3): 304-307, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29364738

RESUMO

PURPOSE: To identify the CRYBA1/A3 mutation spectrum and analyze the genotype-phenotype correlations in Chinese families with congenital cataract. METHODS: Family history and clinical data of 47 unrelated families with autosomal dominant congenital cataract (ADCC) were recorded. CRYBA1/A3 gene sequencing was applied to identify the causative mutation. Haplotypes were constructed using closely linked microsatellite markers and intragenic single-nucleotide polymorphisms (SNPs) to compare the affected haplotype in three families. RESULTS: Nuclear cataract was the most common type of ADCC in Chinese families, accounting for 42.6% (20/47). A recurrent CRYBA1/A3 deletion mutation (ΔG91) was identified in three families (6.4%) with nonprogressive nuclear congenital cataract. Different haplotypes segregated with the mutation in each family. CONCLUSIONS: A recurrent ΔG91CRYBA1/A3 mutation occurs independently in 6.4% of the Chinese families with autosomal dominant nuclear cataracts and most likely represents a mutational hot spot, which underscores the relations between nonprogressive nuclear cataract and CRYBA1/A3.


Assuntos
Catarata/congênito , DNA/genética , Mutação , Cadeia A de beta-Cristalina/genética , Adulto , Catarata/genética , Catarata/metabolismo , Análise Mutacional de DNA , Feminino , Genes Dominantes , Haplótipos , Humanos , Masculino , Linhagem , Recidiva , Cadeia A de beta-Cristalina/metabolismo
11.
Biochem J ; 474(14): 2475-2487, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28592682

RESUMO

Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemized, truncated, and covalently cross-linked. One reaction responsible for age-related protein cross-linking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence for this was found in many lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent cross-links than others. Foremost among them was Cys5 in ßA4 crystallin. The reason for this enhanced reactivity was investigated using peptides. Oxidation of Cys to cystine was a prerequisite for DHA formation, and DHA production was accelerated markedly by the presence of a Lys, one residue separated from Cys5. Modeling and direct investigation of the N-terminal sequence of ßA4 crystallin, as well as a variety of homologous peptides, showed that the epsilon amino group of Lys can promote DHA production by nucleophilic attack on the alpha proton of cystine. Once a DHA residue was generated, it could form intermolecular cross-links with Lys and Cys. In the lens, the most abundant cross-link involved Cys5 of ßA4 crystallin attached via a thioether bond to glutathione. These findings illustrate the potential of Cys and disulfide bonds to act as precursors for irreversible covalent cross-links and the role of nearby amino acids in creating 'hotpsots' for the spontaneous processes responsible for protein degradation in aged tissues.


Assuntos
Cisteína/química , Proteínas do Olho/química , Cristalino/química , Fatores Etários , Alanina/análogos & derivados , Alanina/química , Bases de Dados de Proteínas , Dissulfetos/química , Humanos , Modelos Moleculares , Oligopeptídeos/química , Proteólise , Espectrometria de Massas em Tandem , Cadeia A de beta-Cristalina/química
12.
Eur J Hum Genet ; 25(6): 725-734, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28378818

RESUMO

Infantile nystagmus (IN) is a genetically heterogeneous disorder arising from variants of genes expressed within the developing retina and brain. IN presents a diagnostic challenge and patients often undergo numerous investigations. We aimed to develop and assess the utility of a next-generation sequencing (NGS) panel to enhance the diagnosis of IN. We identified 336 genes associated with IN from the literature and OMIM. NimbleGen Human custom array was used to enrich the target genes and sequencing was performed using HiSeq2000. Using reference genome material (NA12878), we show the sensitivity (98.5%) and specificity (99.9%) of the panel. Fifteen patients with familial IN were sequenced using the panel. Two authors were masked to the clinical diagnosis. We identified variants in 12/15 patients in the following genes: FRMD7 (n=3), CACNA1F (n=2), TYR (n=5), CRYBA1 (n=1) and TYRP1 (n=1). In 9/12 patients, the clinical diagnosis was consistent with the genetic diagnosis. In 3/12 patients, the results from the genetic diagnoses (TYR, CRYBA1 and TYRP1 variants) enabled revision of clinical diagnoses. In 3/15 patients, we were unable to determine a genetic diagnosis. In one patient, copy number variation analysis revealed a FRMD7 deletion. This is the first study establishing the clinical utility of a diagnostic NGS panel for IN. We show that the panel has high sensitivity and specificity. The genetic information from the panel will lead to personalised diagnosis and management of IN and enable accurate genetic counselling. This will allow development of a new clinical care pathway for IN.


Assuntos
Testes Genéticos/métodos , Nistagmo Congênito/genética , Análise de Sequência de DNA/métodos , Adolescente , Adulto , Canais de Cálcio Tipo L/genética , Criança , Pré-Escolar , Proteínas do Citoesqueleto/genética , Feminino , Testes Genéticos/normas , Humanos , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Nistagmo Congênito/diagnóstico , Oxirredutases/genética , Polimorfismo Genético , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas , Cadeia A de beta-Cristalina/genética
13.
Eur J Hum Genet ; 25(6): 711-718, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28272538

RESUMO

Congenital cataract is a rare but severe paediatric visual impediment, often caused by variants in one of several crystallin genes that produce the bulk of structural proteins in the lens. Here we describe a pedigree with autosomal dominant isolated congenital cataract and linkage to the crystallin gene cluster on chromosome 22. No rare single nucleotide variants or short indels were identified by exome sequencing, yet copy number variant analysis revealed a duplication spanning both CRYBB1 and CRYBA4. While the CRYBA4 duplication was complete, the CRYBB1 duplication was not, with the duplicated CRYBB1 product predicted to create a gain of function allele. This association suggests a new genetic mechanism for the development of isolated congenital cataract.


Assuntos
Catarata/genética , Oftalmopatias Hereditárias/genética , Duplicação Gênica , Cadeia A de beta-Cristalina/genética , Cadeia B de beta-Cristalina/genética , Adolescente , Adulto , Idoso , Catarata/patologia , Criança , Pré-Escolar , Cromossomos Humanos Par 22/genética , Variações do Número de Cópias de DNA , Oftalmopatias Hereditárias/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único
14.
Biochem J ; 473(14): 2087-96, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27208166

RESUMO

The high solubility and lifelong stability of crystallins are crucial to the maintenance of lens transparency and optical properties. Numerous crystallin mutations have been linked to congenital cataract, which is one of the leading causes of newborn blindness. Besides cataract, several crystallin mutations have also been linked to syndromes such as congenital microcornea-cataract syndrome (CMCC). However, the molecular mechanism of CMCC caused by crystallin mutations remains elusive. In the present study, we investigated the mechanism of CMCC caused by the X253R mutation in ßB1-crystallin. The exogenously expressed X253R proteins were prone to form p62-negative aggregates in HeLa cells, strongly inhibited cell proliferation and induced cell apoptosis. The intracellular X253R aggregates could be successfully redissolved by lanosterol but not cholesterol. The extra 26 residues at the C-terminus of ßB1-crystallin introduced by the X253R mutation had little impact on ßB1-crystallin structure and stability, but increased ßB1-crystallin hydrophobicity and decreased its solubility. Interestingly, the X253R mutant fully abolished the aggregatory propensity of ßB1- and ßA3/ßB1-crystallins at high temperatures, suggesting that X253R was an aggregation-inhibition mutation of ß-crystallin homomers and heteromers in dilute solutions. Our results suggest that an increase in hydrophobicity and a decrease in solubility might be responsible for cataractogenesis induced by the X253R mutation, while the cytotoxic effect of X253R aggregates might contribute to the defects in ocular development. Our results also highlight that, at least in some cases, the aggregatory propensity in dilute solutions could not fully mimic the behaviours of mutated proteins in the crowded cytoplasm of the cells.


Assuntos
Catarata/genética , Catarata/metabolismo , Doenças da Córnea/genética , Doenças da Córnea/metabolismo , Agregação Patológica de Proteínas/metabolismo , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/metabolismo , Dicroísmo Circular , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutação/genética , Agregação Patológica de Proteínas/genética , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/genética , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/genética
15.
Biochim Biophys Acta ; 1862(6): 1214-27, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26851658

RESUMO

ßγ-Crystallins, having a uniquely stable two domain four Greek key structure, are crucial for transparency of the eye lens,. Mutations in lens crystallins have been proposed to cause cataract formation by a variety of mechanisms most of which involve destabilization of the protein fold. The underlying molecular mechanism for autosomal dominant zonular cataracts with sutural opacities in an Indian family caused by a c.215+1G>A splice mutation in the ßA3/A1-crystallin gene CRYBA1 was elucidated using three transgenic mice models. This mutation causes a splice defect in which the mutant mRNA escapes nonsense mediated decay by skipping both exons 3 and 4. Skipping these exons results in an in-frame deletion of the mRNA and synthesis of an unstable p.Ile33_Ala119del mutant ßA3/A1-crystallin protein. Transgenic expression of mutant ßA3/A1-crystallin but not the wild type protein results in toxicity and abnormalities in the maturation and orientation of differentiating lens fibers in c.97_357del CRYBA1 transgenic mice, leading to a small spherical lens, cataract, and often lens capsule rupture. On a cellular level, the lenses accumulated p.Ile33_Ala119del ßA3/A1-crystallin with resultant activation of the stress signaling pathway - unfolded protein response (UPR) and inhibition of normal protein synthesis, culminating in apoptosis. This highlights the mechanistic contrast between mild mutations that destabilize crystallins and other proteins, resulting in their being bound by the α-crystallins that buffer lens cells against damage by denatured proteins, and severely misfolded proteins that are not bound by α-crystallin but accumulate and have a direct toxic effect on lens cells, resulting in early onset cataracts.


Assuntos
Apoptose , Catarata/genética , Cristalino/patologia , Processamento de RNA , Resposta a Proteínas não Dobradas , Cadeia A de beta-Cristalina/genética , Animais , Sequência de Bases , Catarata/patologia , Linhagem Celular , Éxons , Humanos , Cristalino/citologia , Cristalino/metabolismo , Camundongos Transgênicos , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Deleção de Sequência
16.
Biochim Biophys Acta ; 1860(1 Pt B): 287-98, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26022148

RESUMO

BACKGROUND: Persistent fetal vasculature (PFV) is a human disease in which the fetal vasculature of the eye fails to regress normally. The fetal, or hyaloid, vasculature nourishes the lens and retina during ocular development, subsequently regressing after formation of the retinal vessels. PFV causes serious congenital pathologies and is responsible for as much as 5% of blindness in the United States. SCOPE OF REVIEW: The causes of PFV are poorly understood, however there are a number of animal models in which aspects of the disease are present. One such model results from mutation or elimination of the gene (Cryba1) encoding ßA3/A1-crystallin. In this review we focus on the possible mechanisms whereby loss of functional ßA3/A1-crystallin might lead to PFV. MAJOR CONCLUSIONS: Cryba1 is abundantly expressed in the lens, but is also expressed in certain other ocular cells, including astrocytes. In animal models lacking ßA3/A1-crystallin, astrocyte numbers are increased and they migrate abnormally from the retina to ensheath the persistent hyaloid artery. Evidence is presented that the absence of functional ßA3/A1-crystallin causes failure of the normal acidification of endolysosomal compartments in the astrocytes, leading to impairment of certain critical signaling pathways, including mTOR and Notch/STAT3. GENERAL SIGNIFICANCE: The findings suggest that impaired endolysosomal signaling in ocular astrocytes can cause PFV disease, by adversely affecting the vascular remodeling processes essential to ocular development, including regression of the fetal vasculature. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.


Assuntos
Proteínas do Olho/metabolismo , Vítreo Primário Hiperplásico Persistente/embriologia , Vítreo Primário Hiperplásico Persistente/metabolismo , Vasos Retinianos/anormalidades , Vasos Retinianos/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Animais , Doença Crônica , Humanos , Modelos Biológicos
17.
Acta Med Iran ; 54(12): 778-783, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28120589

RESUMO

Autosomal dominant congenital cataract (ADCC) is the most common form of inherited cataracts and accounts for one-third of congenital cataracts. Heterozygous null mutations in the crystallin genes are the major cause of the ADCC. This study aims to detect the mutational spectrum of four crystallin genes, CRYBA1/A3, CRYBB1, CRYBB2 and CRYGD in an Iranian family. Genomic DNA was isolated from whole blood cells from theproband and other family members. The coding regions and flanking intronicsequences of crystalline genes were analyzed by Sanger sequencing in aproband with ADCC. The identified mutation was further evaluated in available family members. To predict the potential protein partners of CRYBA1/A3, we also used an in-silico analysis. A de novo heterozygous deletion (c.272-274delGAG, p.G91del) in exon 4 of CRYBA1/A3 gene, leading to a deletion of Glycine at codon 91 was found. This genetic variation did not change the reading frame of CRYBA1 protein. In conclusion, we identified a de novo in-frame 3-bp deletion in the proband with an autosomal dominant congenital cataract, but not in her parents, in an Iranian family. This mutation has occurred de novo on a paternal gamete during spermatogenesis. The in-silico results predicted the interaction of CRYBA1 protein with the other CRY as well as proteins responsible for eye cell signaling.


Assuntos
Catarata/genética , Genes Dominantes/genética , Linhagem , Deleção de Sequência/genética , Cadeia A de beta-Cristalina/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Sequência de Bases , Catarata/sangue , Criança , Códon/genética , Análise Mutacional de DNA , Feminino , Variação Genética , Glicina/genética , Humanos , Irã (Geográfico) , Masculino , Dados de Sequência Molecular , Mutação , Pais , Análise de Sequência de DNA/métodos , Cadeia A de beta-Cristalina/sangue , Cadeia B de beta-Cristalina/sangue , Cadeia B de beta-Cristalina/genética , gama-Cristalinas/sangue , gama-Cristalinas/genética
18.
PLoS One ; 10(12): e0144621, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26657544

RESUMO

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and ß-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with ßA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with ßA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for ßA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for ßA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with ßA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ßA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with ßA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor ßA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.


Assuntos
Amidas/metabolismo , Cristalinas/metabolismo , Processamento de Proteína Pós-Traducional , Cadeia B de alfa-Cristalina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalinas/química , Cristalinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cristalino/química , Cristalino/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/genética , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/genética
19.
Int J Biol Macromol ; 76: 86-93, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25709017

RESUMO

The main function of small heat shock proteins acting as suppressors of aggregation of non-native proteins is greatly influenced by crowded environment in the cell and the presence of divalent metal ions. The goal of the present work was to study the effects of Ca(2+) and Mg(2+) ions on the quaternary structure and anti-aggregation activity of αB-crystallin under crowding conditions. We showed that Ca(2+) and Mg(2+) ions induced formation of suboligomeric forms of αB-crystallin. This effect was retained in the presence of crowder (polyethylene glycol), although to a lesser degree. The chaperone-like activity of αB-crystallin was analyzed using heat-induced aggregation of myosin subfragment 1 (S1) at 40°C. In the absence of crowding agents chaperone-like activity of αB-crystallin exhibited low sensitivity to the presence of Ca(2+) and Mg(2+) ions. The addition of the crowding agents (polyethylene glycol 20000, Ficoll 70) dramatically increased S1 aggregation rates and significantly depressed anti-aggregation activity of αB-crystallin. Low concentrations of Ca(2+) (0.1mM) and Mg(2+) (10mM) partially restored the chaperone-like activity of αB-crystallin in the presence of crowders. This effect was observed at relatively low values of [αB-crystallin]/[S1] molar ratio, however, at [αB-crystallin]/[S1]>0.2 the stimulating effect of Ca(2+) became less pronounced. These findings might indicate that under crowded cell conditions different factors, including divalent cations, can effectively modulate chaperone-like activity of protein chaperones, which otherwise cannot properly cope with crowding-provoked accelerated rates of substrates aggregation.


Assuntos
Cálcio/farmacologia , Íons/farmacologia , Magnésio/farmacologia , Multimerização Proteica/efeitos dos fármacos , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/metabolismo , Cálcio/química , Humanos , Magnésio/química , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos
20.
Prog Retin Eye Res ; 44: 62-85, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25461968

RESUMO

Crystallins, the highly abundant proteins of the ocular lens, are essential determinants of the transparency and refractivity required for lens function. Initially thought to be lens-specific and to have evolved as lens proteins, it is now clear that crystallins were recruited to the lens from proteins that existed before lenses evolved. Crystallins are expressed outside of the lens and most have been shown to have cellular functions distinct from their roles as structural elements in the lens. For one major crystallin group, the ß/γ-crystallin superfamily, no such functions have yet been established. We have explored possible functions for the polypeptides (ßA3-and ßA1-crystallins) encoded by Cryba1, one of the 6 ß-crystallin genes, using a spontaneous rat mutant and genetically engineered mouse models. ßA3-and ßA1-crystallins are expressed in retinal astrocytes and retinal pigment epithelial (RPE) cells. In both cell types, these proteins appear to be required for the proper acidification of the lysosomes. In RPE cells, elevated pH in the lysosomes is shown to impair the critical processes of phagocytosis and autophagy, leading to accumulation of undigested cargo in (auto) phagolysosomes. We postulate that this accumulation may cause pathological changes in the cells resembling some of those characteristic of age-related macular degeneration (AMD). Our studies suggest an important regulatory function of ßA3/A1-crystallin in astrocytes. We provide evidence that the cellular function of ßA3/A1-crystallin involves its interaction with V-ATPase, the proton pump responsible for acidification of the endolysosomal system.


Assuntos
Lisossomos/fisiologia , Retina/fisiologia , Cadeia A de beta-Cristalina/fisiologia , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Ratos , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologia
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