Molecular characterization of the glutathione transport system in the lens

晶状体中谷胱甘肽传输系统的分子表征

• 批准号: 9087267
• 负责人: Xingjun Fan
• 金额: $ 23.78万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9087267
• 关键词: Active Biological Transport; Adherent Culture; Affect; Age; Aging; Aging-Related Process; Animal Model; Antioxidants; Breeding; Cataract; Collaborations; Complex; Cysteine; Data; Development; Epithelial; Epithelium; Exploratory/Developmental Grant; Eye; Genes; Genetic; Glutathione; Goals; Health; Homeostasis; Human; Investigation; Knock-out; Laboratories; Ligase; Messenger RNA; Modeling; Molecular; Mus; Nature; Nuclear; Outcome; Oxidation-Reduction; Play; Proteins; Research; Risk Factors; Role; Sequence Analysis; System; Transmembrane Transport; United States; Yeast Model System; Yeasts; age related; deep sequencing; glutathione transporter; innovation; lens; monolayer; oxidation; transcriptome; transcriptome sequencing; yeast genetics; Active Biological Transport; Adherent Culture; Affect; Age; Aging; Aging-Related Process; Animal Model; Antioxidants; Breeding; Cataract; Collaborations; Complex; Cysteine; Data; Development; Epithelial; Epithelium; Exploratory/Developmental Grant; Eye; Genes; Genetic; Glutathione; Goals; Health; Homeostasis; Human; Investigation; Knock-out; Laboratories; Ligase; Messenger RNA; Modeling; Molecular; Mus; Nature; Nuclear; Outcome; Oxidation-Reduction; Play; Proteins; Research; Risk Factors; Role; Sequence Analysis; System; Transmembrane Transport; United States; Yeast Model System; Yeasts; age related; deep sequencing; glutathione transporter; innovation; lens; monolayer; oxidation; transcriptome; transcriptome sequencing; yeast genetics

 DESCRIPTION (provided by applicant): Recent research in my laboratory revealed that conditionally knocking out glutathione (GSH) synthesis from the lens results in a quasi-perfect model of age-related cataractogenesis (the LEGSKO mouse) that simulates most oxidative changes observed in human age-related cataracts. However, subsequent breeding of the mouse was associated with delayed cataractogenesis and a stunning discrepancy between total absence of mRNA and protein for the Gclc subunit of γ-glutamyl cysteine ligase and a persisting 50% (instead of the expected zero%) GSH level in the homozygous LEGSKO mouse, implying thereby the existence of an active transport system for GSH. In preliminary studies I confirmed the ability of the LEGSKO lens to take up H3-GSH against a ten-fold concentration gradient. A preliminary screen revealed that at least 143 candidate transporters from deep sequencing analysis were elevated in the LEGSKO lens out of >1500 changed genes vs. wild type lens. Together, these data provide strong support for the existence of salvage mechanisms implicated in redox and GSH homeostasis in the lens. Yet, previous attempts by others to elucidate the molecular nature of GSH transporter(s) in the lens have failed, implying the presence of a complex problem that I propose to approach using a multipronged, powerful and innovative strategy that combines yeast genetics, transcriptome RNA-seq analysis, a lens epithelial monolayer system specifically developed for transport studies, and the LEGSKO mouse itself. In Specific Aim 1, I will use the yeast model system to perform the initial screen o the candidate transporters selected from the RNA-seq study comparing genes expressed in LEGSKO vs. WT lens. In Specific Aim 2, I will characterize/validate the candidates from yeast screen for GSH transport in lens epithelial monolayer culture system. To achieve these goals I have enlisted the active collaboration an expert in lens genetics (Dr. David Beebe), a yeast geneticist (Dr. Alan Tartakoff) and an expert in epithelium membrane transport mechanisms (Dr. Ulrich Hopfer). I believe the proposed exploratory goals are ideally suited for support by the R21 mechanism, as a successful outcome is expected to open up a vast field of investigation that may altogether have profound implications for redox homeostasis in the aging eye.

 描述（由适用提供）：我的实验室的最新研究表明，从镜头中有条件地敲出谷胱甘肽（GSH）合成导致与年龄相关性白内障发生（legeko小鼠）的准完美模型模拟在与人类年龄相关的白马病中观察到的大多数氧化变化。 However, subsequent breeding of the mouse was associated with delayed cataractogenesis and a stunning discrepancy between total absence of mRNA and protein for the Gclc subunit of γ-glutamyl cysteine ​​ligase and a persisting 50% (instead of the expected zero%) GSH level in the homozygous LEGSKO mouse, implying thereby the existence of an active transport system for GSH.在初步研究中，我证实了Legeko镜头在十倍浓度梯度上占用H3-GSH的能力。初步屏幕表明，在> 1500个更改的基因与野生型镜头中，Legeko镜头中至少有143个来自深层测序分析的候选转运蛋白被提升。总之，这些数据为透镜中氧化还原和GSH稳态实施的打捞机制的存在提供了强有力的支持。然而，其他人以前的尝试阐明镜头中GSH转运蛋白的分子性质已经失败，这意味着我建议使用多重，强大和创新的策略来解决一个复杂的问题，该策略结合了酵母遗传学，转录组RNA-seq分析，透镜上皮单层系统的运输研究，专门为Dembore negs Fornes和Demble Storge Inde and toge nege Fornes和Dembles和Dembore and toge nege and negs Fornes，以及该研究。在特定的目标1中，我将使用酵母模型系统执行初始屏幕o从RNA-Seq研究中选择的候选转运蛋白比较了Legeko与WT镜头中表达的基因。在特定的目标2中，我将表征/验证酵母屏幕中的候选物，用于镜片上皮单层培养系统中的GSH运输。为了实现这些目标，我已经积极合作，是一位镜头遗传学专家（David Beebe博士），酵母遗传学家（Alan Tartakoff博士），并且是上皮膜运输机制（Ulrich Hopfer博士）的专家。我认为，拟议的探险家目标非常适合R21机制，因为预计成功的结果将开辟一个广泛的调查领域，这可能完全对雷克斯体内稳态产生深远的影响。