Molecular characterization of the glutathione transport system in the lens

 描述(申请人提供)：我的实验室最近的研究表明，有条件地敲除晶状体中的谷胱甘肽(GSH)合成会导致一种近乎完美的年龄相关性白内障发生模型(LEGSKO小鼠)，该模型模拟了在人类老年性白内障中观察到的大多数氧化变化。然而，随后繁殖的小鼠与延迟的白内障发生和完全缺乏γ-谷氨酰半胱氨酸连接酶的GClC亚单位的基因和蛋白之间的惊人差异，以及在纯合子的乐高小鼠中持续的50%(而不是预期的0%)谷胱甘肽水平，从而意味着谷胱甘肽存在活跃的运输系统。在初步研究中，我证实了LEGSKO晶状体在十倍浓度梯度下摄取H3-GSH的能力。初步筛选显示，与野生型晶状体相比，LEGSKO晶状体中至少有143个候选转运蛋白在&gt；1500个基因突变的晶状体中升高。总之，这些数据为晶状体中存在与氧化还原和GSH动态平衡有关的挽救机制提供了强有力的支持。然而，之前其他人试图阐明晶状体中谷胱甘肽转运体(S)的分子性质的尝试都失败了，这意味着存在一个复杂的问题，我建议使用一个多管齐下的、强大的和创新的策略来解决这个问题，该策略结合了酵母遗传学、转录组RNA-seq分析、专门为转运体研究开发的晶状体上皮单分子层系统，以及乐高小鼠本身。在具体目标1中，我将使用酵母模型系统对从RNA-SEQ研究中选择的候选转运蛋白进行初步筛选，比较LEGSKO和WT晶状体中表达的基因。在特定的目标2中，我将从酵母筛选中鉴定/验证在晶状体上皮单层培养系统中GSH运输的候选者。为了实现这些目标，我邀请了一位晶状体遗传学专家(David Beebe博士)、一位酵母遗传学家(Alan Tartakoff博士)和一位上皮膜运输机制专家(Ulrich Hopfer博士)积极合作。我相信提出的探索性目标非常适合R21机制的支持，因为一个成功的结果有望打开一个广阔的研究领域，可能总共对老化眼睛的氧化还原动态平衡产生深远的影响。

 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.