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

老化晶状体中谷胱甘肽稳态受损的机制和后果。

• 批准号: 9910409
• 负责人: Xingjun Fan
• 金额: $ 38.41万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-11-10 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910409
• 关键词: Affect; Affinity; Age; Aging; Anabolism; Antioxidants; Binding; Biological; Biological Process; C-terminal; CASP3 gene; Catalytic Domain; Cataract; Cataract Extraction; Cells; Cellular Structures; Collaborations; Competitive Binding; Cytoskeletal Proteins; Cytoskeleton; Dedications; Enzymes; Event; Eye; Fiber; GCLC gene; GCLM gene; Glutathione; Goals; Holoenzymes; Homeostasis; Human; Impairment; Knock-in Mouse; Length; Lens Fiber; Ligase; Link; Molecular; N-terminal; Names; Pathogenesis; Peptide Hydrolases; Protein Subunits; Proteins; Reaction; Research; Resistance; Risk Factors; Role; Structural Models; Testing; Time; Tubulin; United States; age related; design; enzyme activity; enzyme biosynthesis; fiber cell; filamin; in vivo; lens; mouse model; novel; oxidation; ultraviolet irradiation

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!

晶状体谷胱甘肽与年龄相关的下降与人类的白内障形成密切相关 在我们自己的研究中最近证实的谷胱甘肽耗竭的实验小鼠模型中。年龄 - 晶状体谷胱甘肽生物合成酶活性的相关损害已被证明，尽管 这种与年龄相关的变化背后的机制知之甚少。在初步研究中，我们使 范式转移观察，即晶状体谷胱甘肽生物合成酶，γ谷氨酰胺半胱氨酸 连接酶，催化亚基（GCLC）经过翻译后截断，并且该截短的蛋白质成型 随着年龄的增长，特别是在晶状体皮质和核纤维区域中积累。进一步的研究表明 截断与caspase-3和6的存在相关，如裂解基序，触发C末端13KD N末端60kD稳定蛋白质成型型的截断和积累（此处称为GCLC60）。后者出现 与细胞细胞骨架蛋白和深层细胞质分布进行牢固的相互作用 与全长GCLC蛋白相比，差异。我们假设GCLC的截断是一个关键事件 与年龄相关白内障的发病机理，以及理解GCLC60和 其积累的分子生物学后果是设计新型抗狂欢的重要目标 治疗。因此，本应用的三个特定目的是目标1：确定GCLC的机制 截断及其对镜头GSH稳态的影响。特别是，我们将测试GCLC60的假设 抑制GSH合成，导致GSH含量降低，并将确定 负责与年龄有关的裂解。在AIM 2中，我们将确定体内对GCLC截断的影响 GCLC60通过测试抗切割抗蛋白（KI）小鼠模型来积累。特别是我们假设 Ki小鼠透镜将在衰老过程中显着保留其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引起的生物学作用，假设是 GCLC积累导致细胞骨架基质混乱。 呢1！