先天性白内障相关的βB2晶状体蛋白突变对其结构、稳定性及蛋白相互作用的影响

βB2-Crystallin is one of major protein constituents of the human lens, its high solubility and stability are critical for lens clarity and refraction. Well recognized as a structural protein, other physiologic functions of βB2-crystallin remains unkownn. Many congenital cataract-linked mutations in βB2-crystallin gene (CRYBB2) have been reported. However, researchs focus on pathopoiesis mechanism of these mutations are still rare. Here we base on the mutations that are already published, use site-directed mutagenesis to constuct plasmids, and product recombinant proteins by overexpressing them in E.coli. Then the effect of mutations on βB2-crystallin structure and stability is investigated by biophysical methods, such as circular dichroism, fluorescence and X-ray crystal diffraction. Meanwhile, we transfect HeLa cells with the plasmids containing coding sequence of wild or mutant βB2-crystallin, for observing subcellular localization of target proteins, also for analyzing the effcet of the mutant proteins on cell proliferation and apoptosis. Moreover, we plan to study the protein-protein interantions by yeast two-hybird system, CO-IP and FRET methods. During our research, through elucidating pathopoiesis mechanism of these congenital cataract-linked mutations at both protein level and cell level, we can get a better understanding of the relationship between protein alteration and cataractogenesis, as well as creating a foundation which will help us in further studying the physiologic functions of βB2-crystallin and gene therapy of congenital catatact.

βB2晶状体蛋白是人眼晶状体中重要的结构蛋白，与维持晶状体的透明性与屈光性密切相关，其潜在的生理功能尚不明确。目前国内外针对βB2晶状体蛋白基因突变导致先天性白内障的报道已有不少，但是针对突变具体的致病机制研究仍很缺乏。本课题拟从现有的与βB2晶状体蛋白相关的基因突变中，选取有意义的关键位点，构建野生型βB2晶状体蛋白和包含各突变位点的质粒；通过原核表达获取目标蛋白，并明确其突变前后结构和理化性质方面改变；转染真核细胞，观察蛋白在细胞内定位以及对细胞生理状态的影响；同时用多种方式研究βB2晶状体蛋白与其他蛋白的相互作用，结合突变来探索其相互作用位点。本课题拟分别在蛋白水平和细胞水平，较完整地揭示这些先天性白内障相关基因突变对βB2晶状体蛋白结构、稳定性和蛋白相互作用等方面的影响，从而更加明确白内障的发生机理，为进一步研究βB2的生理功能打下基础，也为先天性白内障的防治提供理论依据。

本研究根据预期研究方案，从分子生物学和生物物理学角度出发，在目前已发现的βB2晶状体蛋白基因突变中选取βB2晶状体蛋白C末端最后一个折叠片层的两个关键位点上的三个突变（V187M、V187E和R188H）以及两个位于蛋白结构域疏水核心的保守色氨酸突变（W59C和W151C）,在结构上和功能上对其相应的致病机制进行研究。研究在体外大量表达蛋白并纯化，运用生物物理学手段如圆二色谱、核磁共振、荧光光谱、电镜等方法检测突变蛋白的结构和理化性质；同时将突变蛋白的基因转染到真核细胞中，观察其亚细胞定位、细胞毒性以及与其他蛋白质的相互作用。研究发现βB2晶状体蛋白C末端最后一个折叠片层V187、R188位点及蛋白结构域疏水核心的保守色氨酸位点W59、W151在βB2晶状体蛋白结构、稳定与折叠中起到了关键性作用，揭示了这些位点突变引起先天性白内障的分子机制，为进一步研究βB2的生理功能打下基础，也为先天性白内障的防治提供理论依据。