α晶状体蛋白外消旋化在糖尿病性白内障发病中的作用机制研究

Diabetic cataract (DC) is a blinding type of cataract featuring early onset, great operative difficulty, various complicating vitreoretinopathy and many postsurgical complications but with unclear pathogenesis. Previous studies have found post-translational modification (PTM) of crystallines was one of the most important clues in lens opacification, and racemisation had the highest level among numbers of PTMs. Alpha-crystallin is the most important structural protein of humanbeing, and we previously found its role in stabilization of cell membrane besides its function as a molecular chaperone of other crystallines. We suspect that racemisation of alpha-crystallin played a role in pathogenesis of diabetic cataract by protein denaturation. In this project, we plan to analyse the influence of alpha-crystallin racemisation in pathogenesis of diabetic cataract though two main aspects, change of crystallins and fluidity of membrane. Firstly, we will use revised phase HPLC technique to analyse the location and level of alpha-crystallin racmisation in diabetic cataract patients and rat models. Then we will analyze the function of alpha-crystallin of patients and rat models as molecular chaperone and in stabilization of membrane. At last, we will futher analyze the function of alpha-crystallin in the two aspects described above after reversing its racmisation using protein L-isoaspartyl methyltransferase (PIMT) in lenses of DC patients and rat models. We hope this project could provide a new direction for prevention and treatment of diabetic cataract in the future.

糖尿病性白内障（DC）是高致盲风险的白内障类型，具有发病年龄早，手术难度大，眼底病变及术后并发症常见等特点，但目前发病机制不明。前期研究表明晶状体变性的重要线索之一是晶状体蛋白翻译后修饰（PTM），其中含量最高的是外消旋化。α晶状体蛋白是人晶状体中最重要的结构蛋白，除外对晶状体蛋白的分子伴侣作用，我们前期还发现了其具有稳定晶状体细胞膜的作用，而外消旋化可能导致其变性沉淀从而显著影响DC的发生和发展。本课题将从晶状体蛋白和细胞膜流动性改变入手，分析α晶状体蛋白外消旋化在DC发病中的作用。首先利用反相HPLC技术，分析糖尿病性白内障患者及动物模型中，α晶状体蛋白外消旋化的位点和程度，再观察其分子伴侣功能及对晶状体细胞膜的稳定效应；然后进一步用L-异天冬氨酰甲基转移酶（PIMT）特异性地逆转α晶状体蛋白Asp残基的外消旋化后观察其效应。通过本研究希望为糖尿病性白内障的防治提供新方向。

糖尿病性白内障（DC）是高致盲风险的白内障类型，具有发病年龄早，手术难度大，眼底病变及术后并发症常见等特点，但目前发病机制不明。前期研究表明晶状体变性的重要线索之一是晶状体蛋白翻译后修饰（PTM），其中含量最高的是外消旋化。α晶状体蛋白是人晶状体中最重要的结构蛋白，而外消旋化可能导致其变性沉淀从而显著影响DC的发生和发展。本课题从晶状体蛋白入手，分析α晶状体蛋白外消旋化在DC发病中的作用。首先利用反相HPLC技术，分析糖尿病性白内障患者中，α晶状体蛋白外消旋化的位点和程度，再观察其分子伴侣功能；然后进一步用L-异天冬氨酰甲基转移酶（PIMT）特异性地逆转α晶状体蛋白Asp残基的外消旋化后观察其效应。我们发现DC患者第58位Asp残基D/L同分异构体比例差异与对照年龄相关性白内障（ARC）组有统计学意义，αA晶状体蛋白中保护性的左旋L-Asp 58 向有害的右旋D-Asp 58异构体的转化显著增加，可能是导致DC临床进展更迅速的原因。进一步区分皮质及核区组织的检测分析显示，晶状体样本皮质中D-Asp 58比例在DC患者显著高于对照组，同时L-Asp的比例显著降低，提示DC患者临床表型与ARC不同的原因。且随着糖尿病病程延长核区的D-isoAsp 58比例显著增加，可溶性蛋白比例显著下降，α晶状体蛋白分子伴侣功能下降，导致白内障早发。经PIMT逆转后α晶状体蛋白外消旋化程度降低，分子伴侣功能提升，提示α晶状体蛋白外消旋化在DC发病中发挥重要作用，可能成为未来药物治疗靶点。