Dataset Information

Identification of proteins that modify cataract of mouse eye lens.

ABSTRACT: The occurrence of a nuclear cataract in the eye lens due to disruption of the alpha3Cx46 connexin gene, Gja3, is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, differences in gene expression and PTMs occurring during pathology and between individuals. The abundance of 63 protein species was altered between the strains. A unique aspect of this study is the identification of chaperonin subunit 6A, mortalin, ERp29, and syntaxin-binding protein 6 in the eye lens. DNA polymorphisms resulting in nonconservative amino acid changes that led to altered physicochemical properties of the proteins were detected for mortalin, chaperonin subunit 6A, annexin A1, and possibly gamma-N crystallin. The results show HSP27/25 and/or ERp29 are the likely major modifying factors for cataractogenesis. Extension of the results suggests that small heat-shock proteins have a major role for influencing cataract formation in humans.

SUBMITTER: Hoehenwarter W

PROVIDER: S-EPMC3018240 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Identification of proteins that modify cataract of mouse eye lens.

Hoehenwarter Wolfgang W Tang Yajun Y Ackermann Renate R Pleissner Klaus-Peter KP Schmid Monika M Stein Robert R Zimny-Arndt Ursula U Kumar Nalin M NM Jungblut Peter R PR

Proteomics 20081201 23-24

The occurrence of a nuclear cataract in the eye lens due to disruption of the alpha3Cx46 connexin gene, Gja3, is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, diff ...[more]

PMID: 19003866

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Project description:The primary energy substrate of the lens is glucose and uptake of glucose from the aqueous humor is dependent on glucose transporters. GLUT1, the facilitated glucose transporter encoded by Slc2a1 is expressed in the epithelium of bovine, human and rat lenses. In the current study, we examined the expression of GLUT1 in the mouse lens and determined its role in maintaining lens transparency by studying effects of postnatal deletion of Slc2a1. In situ hybridization and immunofluorescence labeling were used to determine the expression and subcellular distribution of GLUT1 in the lens. Slc2a1 was knocked out of the lens epithelium by crossing transgenic mice expressing Cre recombinase under control of the GFAP promoter with Slc2a1loxP/loxP mice to generate Slc2a1loxP/loxP;GFAP-Cre+/0 (LensΔGlut1) mice. LensΔGlut1 mice developed visible lens opacities by around 3 months of age, which corresponded temporally with the total loss of detectable GLUT1 expression in the lens. Spectral domain optical coherence tomography (SD-OCT) imaging was used to monitor the formation of cataracts over time. SD-OCT imaging revealed that small nuclear cataracts were first apparent in the lenses of LensΔGlut1 mice beginning at about 2.7 months of age. Longitudinal SD-OCT imaging of LensΔGlut1 mice revealed disruption of mature secondary fiber cells after 3 months of age. Histological sections of eyes from LensΔGlut1 mice confirmed the disruption of the secondary fiber cells. The structural changes were most pronounced in fiber cells that had lost their organelles. In contrast, the histology of the lens epithelium in these mice appeared normal. Lactate and ATP were measured in lenses from LensΔGlut1 and control mice at 2 and 3 months of age. At 2 months of age, when GLUT1 was still detectable in the lens epithelium, albeit at low levels, the amount of lactate and ATP were not significantly different from controls. However, in lenses isolated from 3-month-old LensΔGlut1 mice, when GLUT1 was no longer detectable, levels of lactate and ATP were 50% lower than controls. Our findings demonstrate that in vivo, the transparency of mature lens fiber cells was dependent on glycolysis for ATP and the loss of GLUT1 transporters led to cataract formation. In contrast, lens epithelium and cortical fiber cells have mitochondria and could utilize other substrates to support their anabolic and catabolic needs.

Project description:PURPOSE:We retrospectively assessed the incidence of cataracts in patients with retinoblastoma (Rb) treated with either lens-sparing radiation therapy (LSRT) or whole-eye radiation therapy (WERT). A secondary aim of this study was to model the dose-response risk of cataract. METHODS AND MATERIALS:We reviewed 65 patients with Rb treated with radiation therapy (RT) at Children's Hospital, Los Angeles from 1997 to 2015. Eyes that were enucleated before RT or lacked follow-up eye examinations were excluded. All patients underwent computed tomography simulation, and mean lens dose data were collected. Follow-up ophthalmologic examinations and intraocular lens implant history were reviewed for cataracts. The primary event-free survival (EFS) outcome was cataract development. Eyes without cataracts were censored on the last date of eye examination or post-RT enucleation, if applicable. Kaplan-Meier estimates were used to compare EFS outcomes, and dose response was projected with Cox regression and logistic regression models. RESULTS:Sixty-one patients (94 eyes) were analyzed with a median follow-up of 51.8 months. For eyes treated with WERT, cataracts developed in 71.7% versus 35.3% for LSRT. Median EFS for WERT and LSRT were 20.8 and 67.9 months, respectively. Compared with WERT, a significant EFS benefit was demonstrated for LSRT (P < .001). Mean lens dose had a significant effect on cataracts in both Cox regression and logistic regression models (P < .01). The mean lens dose of 7 Gy was projected to have a 5-year cataract incidence of 20% and 25% with the logistic and Cox regression models, respectively. CONCLUSIONS:We report the first clinical data demonstrating significantly improved EFS in patients with Rb treated with LSRT. Through lens dose-response modeling, we validate a mean lens dose threshold of 7 Gy to keep cataract risk below 25%. Although RT is used less often for Rb owing to advances in chemotherapy delivery options, these findings are relevant for refining lens dose constraints, particularly in children who have received radiation dose near the orbit.

Project description:PURPOSE: Phosphorylation is an important post-translational modification for the cellular regulation of various biosignaling pathways. We have identified in vivo phosphorylation sites of various lens proteins including especially the major structural proteins of the crystallin family from porcine eye lenses by means of two-dimensional gel electrophoresis (2-DE) or immobilized metal affinity chromatography (IMAC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). METHODS: For the identification of phosphorylated residues in various lens proteins of porcine lens extracts, we have adapted two complementary proteomic approaches, i.e., pre-fractionation of protein samples with 2-DE or enrichment of phosphopeptides with IMAC followed by LC-MS/MS analysis and database search. The results were compared and validated with those in phosphoproteomics databases. RESULTS: Two subunits of alpha-crystallin, alphaA-crystallin and alphaB-crystallin, as well as other lens crystallins and non-crystallin cellular proteins, such as beta-enolase, heat shock protein beta-1 (HSP27), and glucose-6-phosphate isomerase (GPI) were found to be phosphorylated in vivo at specific sites. Moreover, alphaA- and alphaB-crystallins were found to be the most abundantly phosphorylated proteins in porcine lenses, being extensively phosphorylated on serine or threonine, but not on tyrosine residues. CONCLUSIONS: The complementary gel-based and gel-free proteomic strategies have been compared and evaluated for the study of crystallin phosphorylation from whole tissue extracts of porcine eye lenses. Technically, the IMAC method facilitates direct site-specific identification of phosphorylation residues in lens proteins, which does not necessitate the pre-MS/MS 2-DE separation of protein samples. Moreover, the improved strategy using gel-free phosphoproteomics analysis affords a more effective and simplistic method for the determination of in vivo phosphorylation sites than the conventional 2-DE pre-separation of protein mixture. This study should form a firm basis for the comprehensive analysis of post-translational modification of lens proteins in terms of aging or various diseased states.

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Identification of proteins that modify cataract of mouse eye lens.

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Identification of proteins that modify cataract of mouse eye lens.

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