Molecular mechanisms of protein crosslinking in the lens

透镜蛋白在老化过程中经历二硫键和非二硫键交联。这样的交联是 与蛋白质聚集、不溶解、光散射和透镜调节丧失有关。而 二硫键交联研究得很好，非二硫键的生物化学性质和形成机制 对交联的了解很少。我们建议测试一个新的假设，即非二硫键共价键 透镜中蛋白质的交联可能由复合物的糖基化介导的交联引起， α-晶状体蛋白与其伴侣蛋白之间形成的蛋白质， 在透镜老化过程中，蛋白质的重量和蛋白质的不溶。我们的初步研究强烈支持这一点 假说.在这个项目中，我们将通过三个具体目标系统地研究这个假设。 在目标1中，我们将进行实验以建立α-stac-客户蛋白的长期稳定性 复合物的条件下的透镜，通过采用荧光共振能量转移（FRET）- 基于分析。然后，我们将确定α-堆叠-客户蛋白复合物是否经历更多的共价键， 通过量化蛋白质交联， 晚期糖基化终产物（AGEs）。在目标2中，我们将把我们的研究扩展到完整的人类和小鼠。 镜片，以确定是否氧化或热应激（以促进α-堆叠-客户端蛋白复合物 形成）将促进透镜中糖基化介导的蛋白质交联。然后我们将确定 应力和糖化对镜片透光率和硬度（弹性）的集体影响。在目标3中， 我们将首先确定α-stac-客户蛋白糖基化交联是否与 非二硫键交联的高分子量蛋白质在老化的晶状体;然后，我们将使用一种新的抑制剂 我们在上一个资助期开发的抑制人类晶状体中蛋白质交联的药物。最后， 我们将确定该抑制剂是否防止了透光率的损失和弹性的损失， AGE介导的蛋白质交联。这三个目标将共同测试蛋白质的创新概念 在透镜中的交联，并测试针对这种交联的新型化学抑制剂。本研究结果 可能会带来治疗老花眼和白内障的创新疗法。

Lens proteins undergo disulfide and non-disulfide crosslinking during aging. Such crosslinkings are associated with protein aggregation, insolubilization, light scattering and loss of lens accommodation. While disulfide crosslinking is well studied, the biochemical nature and the mechanism of formation of non-disulfide crosslinking are poorly understood. We propose to test a novel hypothesis that non-disulfide covalent crosslinking of proteins in the lens could arise from glycation-mediated crosslinking of the complexes that are formed between α-crystallin and its chaperoned proteins, which leads to the formation of high molecular weight proteins and protein insolubilization during lens aging. Our preliminary studies strongly support this hypothesis. In the proposed project, we will systematically investigate this hypothesis via three specific aims. In Aim 1, we will perform experiments to establish the long-term stability of α-crystallin-client protein complexes under the conditions of the lens by employing fluorescence resonance energy transfer (FRET)- based assays. We will then determine whether α-crystallin-client protein complexes undergo more covalent crosslinking by glycation than their individual protein components by quantifying protein-crosslinking advanced glycation end products (AGEs). In Aim 2, we will extend our studies to intact human and mouse lenses to determine whether oxidative or thermal stress (to promote α-crystallin-client protein complex formation) would promote glycation-mediated protein crosslinking in the lens. We will then determine the collective effects of stress and glycation on light transmittance and stiffness (resilience) in lenses. In Aim 3, we will first determine whether crosslinking by α-crystallin-client protein glycation has a direct relationship with non-disulfide crosslinked high-molecular-weight proteins in aging lenses; we will then use a novel inhibitor that we developed during the previous funding period to inhibit protein crosslinking in human lenses. Finally, we will determine whether the inhibitor prevents losses in light transmittance and losses in resilience due to AGE-mediated protein crosslinking. Together, the three aims will test an innovative concept of protein crosslinking in the lens and test a novel chemical inhibitor against such crosslinking. The findings in this study could lead to innovative therapies against presbyopia and cataracts.