Molecular mechanisms of protein crosslinking in the lens

晶状体蛋白质在老化过程中经历二硫键和非二硫键的交联化。这样的交叉连接是 与蛋白质聚集、不溶解、光散射和晶状体调节丧失有关。而当 对二硫键的交联性、非二硫键的生化性质和形成机理进行了深入的研究 人们对交联剂知之甚少。我们建议检验一个新的假设，即非二硫键共价 晶状体中蛋白质的交联可能是由糖基化介导的复合体的交联引起的 α-晶体蛋白与其伴侣蛋白之间形成的高分子结构 重量蛋白质和晶状体老化过程中的蛋白质不溶解。我们的初步研究有力地支持了这一点 假设。在拟议的项目中，我们将通过三个具体目标系统地研究这一假说。 在目标1中，我们将进行实验以确定α-晶体蛋白-客户蛋白的长期稳定性 利用荧光共振能量转移(FRET)在晶状体条件下形成的络合物- 基础化验。然后我们将确定α-晶体蛋白-客户蛋白复合体是否经历更多的共价 糖基化交联法比定量蛋白质交联法测定单个蛋白质组分的交联度 晚期糖基化终末产物(AGEs)。在目标2中，我们将把我们的研究扩展到完整的人和小鼠 确定氧化或热应激(促进α-晶体蛋白-客户蛋白复合体)的镜片 形成)将促进糖基化介导的蛋白质在晶状体中的交联。然后我们将确定 压力和糖基化对晶状体透光率和硬度(弹性)的共同影响。在《目标3》中， 我们将首先确定通过α-晶体蛋白-客户蛋白糖基化进行的交联是否与 老化晶状体中的非二硫交联型高分子量蛋白质；然后我们将使用一种新的抑制剂 这是我们在之前的资助期间开发的，用于抑制人类晶状体中的蛋白质交联。最后， 我们将确定该抑制剂是否可以防止由于以下原因造成的透光率损失和弹性损失 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 在晶状体中进行交联，并测试一种新型的化学抑制剂以防止这种交联。这项研究的发现 可能会导致针对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.