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ABSTRACT:
PROVIDER: PRJNA1105232 | ENA |
REPOSITORIES: ENA
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Project description:The alpha-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataract, the leading cause of human blindness. There are conflicting data in the literature as to what role that alpha-crystallins may play in early lens development. In this study we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three alpha-crystallin genes (cryaa, cryaba and cryabb). Absence of protein was confirmed by mass spectrometry and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of alpha A-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf, but little significant change in normal fiber cell denucleation. Loss of either Alpha Ba- or Alpha Bb-crystallin produced no significant lens defects. Mutation of each Alpha-crystallin gene did not alter the expression levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for Alpha A-crystallin in lens development, but the range of phenotype severity suggests its loss simply increases the chance for defect, and that the protein is not essential. Our finding that cryaba and cryabb null mutants lack noticeable lens defects is congruent with insignificant transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.
2022-01-20 | PXD030918 | panorama
Project description:Age-related breakdown of lenticular crystallins is associated with lenticular disorders such as cataract. Despite playing a critical role in maintaining lens homeostasis, the mechanism(s) and consequences of this phenomenon are not well understood. Utilising a proteomic-based approach, this study characterised 238 endogenous peptides derived from age-related crystallin breakdowns present in the cortical tissues of young, middle-age and old human lenses. Quantitative mass spectrometry analysis showed that the concentration of a prominent crystallin breakdown product in the lens increased significantly with age, which, coupled with the age-related increase in variety of the LMW crystallin peptide in the lens cortex, suggests that a major crystallin breakdown event taking place in the human lens cortex shortly after middle-age. In-depth analysis on the crystallin peptide terminal amino acids indicate the presence of trypsin-like proteolysis in the lens cortical cells, providing useful information on the mechanism(s) that contribute to crystallin breakdown in the aging human lens. Taken together, this work enhances our understanding on the age-related crystallin breakdown process in the cortical tissues of the human lens.
2016-09-29 | PXD001630 | Pride
Project description:Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract. Microarray comparision of lenses from mixed background (129Sv/J, C57BL/6J, and ICR) control (MafG+/-:MafK+/-; no-cataract) and compound (MafG-/-:MafK+/-; cataract) mouse mutants
2015-05-01 | E-GEOD-65500 | biostudies-arrayexpress
Project description:Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract.
2015-05-01 | GSE65500 | GEO
Project description:For studying the pathogenesis of glucocorticoid induced cataract, We established stable overexpression of αA- and αB-crystallin in human lens epithelial cells (HLECs). All cells were treated with 0.1 μmol, 1 μmol, 10 μmol and 100 μmol Dex for 12 h, 24 h, 36 h, and 48 h then we detected closely related lncRNAs. Our results showed that αA-crystallin promoted the expression of some anti-apoptotic proteins. Lnc-TMPRSS15 played a very important role which provides a theoretical basis for the mechanism of glucocorticoid-induced cataract.
2022-01-16 | GSE193629 | GEO
Project description:A large variety of low molecular weight (LMW) peptides, derived from the breakdown of crystallins, have been reported in middle to old age human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilisation of crystallins – these being hall marks of cataractogenesis. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of human lenses aged 16, 44, 75 and 83 years. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C-terminus with significantly higher frequency compared to other residues, suggesting that trypsin-like proteolysis may be active in the lens cortical fibre cells. Selected reaction monitoring (SRM) analysis of a prominent αA-crystallin peptide (αA57-65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA57-65 formation escalated significantly. Using 2-D gel electrophoresis and nanoLC-ESI-MS/MS, 13 protein complexes of 40-150 kDa consisting of multiple crystallin components were characterised from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross-linking, with these complexes potentially acting to stabilise degraded crystallins by sequestration into water soluble complexes.
2016-09-29 | PXD001593 | Pride
Project description:Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract (ARNC). To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- vs. intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ~4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H2O2, while γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33 and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130 and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose C42, C79/80, C154 in γC; C42, C33 in γD, and C83, C115 and C130 in γS. In conclusion, while the β-crystallin disulfidome is highly conserved in ARNC and LEGSKO mouse, and reproducible by in vitro oxidation, some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of ARNC.
2015-10-16 | PXD002658 | Pride
Project description:Transcriptomic analysis of human lens and age-related cataract
| PRJNA881318 | ENA
Project description:To explore the regulatory mechanism of age-related cataract (ARC) formation and progression，we construct sodium selenite-induced rat cataract model and performed the high-throughput RNA sequencing (HTS) technology to identify the mRNA and miRNA expression profiles of the lens from Na2Se03-induced and saline - injected Sprague Dawley rats.
2023-11-22 | GSE226273 | GEO
Project description:To explore the regulatory mechanism of age-related cataract (ARC) formation and progression，we construct sodium selenite-induced rat cataract model and performed the high-throughput RNA sequencing (HTS) technology to identify the mRNA and miRNA expression profiles of the lens from Na2Se03-induced and saline - injected Sprague Dawley rats.
2023-11-22 | GSE226272 | GEO