Mechanisms and consequences of impaired glutathione homeostasis in the aging Lens.

晶状体谷胱甘肽的年龄相关性下降与人类白内障的形成密切相关 以及实验小鼠的谷胱甘肽耗竭模型，最近我们自己的研究证实了这一点。年龄- 晶状体谷胱甘肽生物合成酶活性的相关损害已被记录在案，尽管 这种与年龄相关的变化背后的机制还知之甚少。在初步研究中，我们做出了 晶状体谷胱甘肽生物合成关键酶--谷氨酰半胱氨酸的范式转换观察 连接酶催化亚基(GCLC)受到翻译后截断的影响，这种截断的蛋白质形式 随着年龄的增长而积累，特别是在晶状体皮质和核心纤维区。进一步的研究表明， 截断与触发C-末端13kD的类似caspase-3和6的切割基序的存在有关 截断和积累一个N-末端60kD的稳定蛋白形式(这里称为Gclc60)。后者出现了 与细胞骨架蛋白有很强的相互作用，细胞质分布广泛 与全长GCLC蛋白相比有差异。我们假设GCLC的截断是 老年性白内障的发病机制，以及对Gclc60和Gclc60形成机制的理解 其蓄积的分子生物学后果是设计新型抗白内障药物的重要目标 心理治疗。因此，本申请的三个具体目的是目标1：确定GCLC的机制 截断及其对晶状体GSH动态平衡的影响特别是，我们将测试Gclc60将 抑制GSH合成，导致GSH含量降低，并将识别 负责与年龄相关的乳沟。在目标2中，我们将确定体内对GCLC截断和 通过测试抗切割敲打(KI)小鼠模型来检测Gclc60的积聚。特别是，我们假设 KI小鼠晶状体在老化过程中将显著保持其GSH水平和生物学功能。在Aim3中，我们将 确定Gclc60与细胞骨架蛋白相互作用所产生的生物效应，假设 GCLC积聚导致细胞骨架基质解体。 ！1！

The age-related decline of lens glutathione is strongly correlated with cataract formation in the human and in experimental mouse models of glutathione depletion, as recently confirmed in our own studies. The age- related impairment of lens glutathione biosynthesis enzyme activity has been documented, though the mechanisms underlying this age-related change are poorly understood. In preliminary studies we made the paradigm shifting observation that the lens glutathione biosynthesis key enzyme, gamma glutamylcysteine ligase, catalytic subunit (Gclc) is subject to posttranslational truncation, and that this truncated proteoform accumulates with age, particularly in the lens cortical and core fiber region. Further studies revealed that the truncation is linked to the presence of a caspase-3 and 6 like cleavage motif triggering a C-terminal 13kD truncation and accumulation of a N-terminal 60kD stable proteoform (herein named Gclc60). The latter appears to engage in strong interaction with cell cytoskeleton proteins and profound cytoplasmic distribution discrepancies compared to the full length Gclc protein. We hypothesize that truncation of Gclc is a key event in the pathogenesis of age-related cataract, and that understanding the mechanism of formation of Gclc60 and the molecular biological consequences of its accumulation are important goals for the design of novel anti-cataract therapy. Accordingly, the three Specific Aims of this application are Aim 1: to determine the mechanism of Gclc truncation and its impact on lens GSH homeostasis. In particular, we will test the hypothesis that Gclc60 will suppresses GSH synthesis resulting in lowered GSH content and will identify the proteases that are responsible for age-related cleavage. In Aim 2, we will determine the in vivo effects on GCLC truncation and Gclc60 accumulation by testing a cleavage resistant knockin (KI) mouse model. In particular we hypothesize that KI mouse lens will significantly retain its GSH levels and biological functions during aging. In Aim3, we will determine the biological effects resulting from Gclc60 interaction with cytoskeletal proteins, hypothesizing that Gclc accumulation results in cytoskeletal matrix disorganization. ! 1!