Molecular characterization of the glutathione transport system in the lens

 描述（由申请人提供）：我实验室的最新研究显示，有条件地敲除透镜中谷胱甘肽（GSH）的合成可产生年龄相关性白内障发生的准完美模型（LEGSKO小鼠），该模型模拟了在人类年龄相关性白内障中观察到的大多数氧化变化。然而，小鼠的后续繁殖与延迟的白内障发生相关，并且在纯合子LEGSKO小鼠中完全缺乏γ-谷氨酰半胱氨酸连接酶Gclc亚基的mRNA和蛋白质与持续50%（而不是预期的0%）GSH水平之间存在惊人的差异，从而暗示存在GSH的主动转运系统。在初步研究中，我证实了LEGSKO透镜在10倍浓度梯度下吸收H3-GSH的能力。初步筛选显示，与野生型透镜相比，在>1500个改变的基因中，来自深度测序分析的至少143个候选转运蛋白在LEGSKO透镜中升高。总之，这些数据为透镜中存在涉及氧化还原和GSH稳态的补救机制提供了强有力的支持。然而，其他人先前试图阐明透镜中GSH转运蛋白的分子性质的尝试失败了，这意味着存在一个复杂的问题，我建议使用多管齐下的，强大的和创新的策略，结合酵母遗传学，转录组RNA测序分析，专门为运输研究开发的透镜上皮单层系统，和LEGSKO小鼠本身。 在具体目标1中，我将使用酵母模型系统对从比较LEGSKO与WT透镜中表达的基因的RNA-seq研究中选择的候选转运蛋白进行初始筛选。在具体目标2中，我将表征/验证来自酵母筛选的候选物在透镜上皮单层培养系统中的GSH转运。为了实现这些目标，我已经招募了一位透镜遗传学专家（大卫毕比博士）、一位酵母遗传学家（艾伦·塔尔塔科夫博士）和一位上皮细胞膜转运机制专家（乌尔里希·霍普费尔博士）的积极合作。我相信所提出的探索性目标非常适合R21机制的支持，因为成功的结果有望开辟一个广阔的研究领域，这可能对衰老眼睛的氧化还原稳态产生深远的影响。

 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.