紫外线或氧化应激状态下α-晶状体蛋白在晶状体内的保护调控机理研究

Cataract, opacification of the lens, is one of the commonest causes of loss of useful vision. The lens will be subjected to various external stimuli with age, such as ultraviolet radiation and oxidative stress. Protein aggregation caused by ultraviolet radiation is one of the pathogenic factors of age-related cataract. Trauma, surgery, systemic metabolic diseases, drugs, excessive drinking and smoking will cause the oxidative damage and biochemical changes of lens cell, resulting in accumulation of misfolding or insoluble protein, leading to cataracts. The α-crystallin (α-CRY) family, which has the highest content in the mammals lens, has a molecular chaperone function and plays an important role in helping irreversible proteins misfolding correctly and to maintain lens transparency. Thus, regulation of chaperone activity of α-CRY is the key target for therapy of cataract. Previous studies have found that α-CRY can form a regular intracellular body (α-CRY body) under ultraviolet radiation and oxidative stress, and it is hypothesized that α-CRY body plays a certain role in maintaining the lens homeostasis. The project aims to isolate and purify the α-CRY body by multi-disciplinary techniques such as nuclear separation, immunoprecipitation and cellular flow sorting, then further analyze its molecular structure and components. And on this basis to describe the mechanism and physiological functions of α-CRY body under ultraviolet radiation and oxidative stress. At the same time, the project plans to screen some natural compounds which can activate or inhibit the formation of α-CRY body, providing a new strategy for the precise prevention and treatment of cataract.

白内障是全球致盲率最高的眼科疾病。紫外线辐射、晶状体氧化损伤，造成晶状体内蛋白质损伤累积，产生聚集甚至沉淀，是导致白内障发生的主要致病因素。作为哺乳动物晶状体内含量最高的α-晶状体蛋白(α-CRY)家族，具有分子伴侣功能，辅助其他晶状体蛋白正确折叠，在维持眼球屈光性中发挥重要功能。因此，调控α-CRY分子伴侣活性是白内障靶向治疗的关键方案。本项目前期研究发现在紫外线辐射、氧化应激条件下，α-CRY形成规则的细胞内小体(α-CRY body)，猜测α-CRY body在维持晶状体稳态发挥特殊功能。本项目旨在通过生物物理学、细胞生物学等多学科技术手段分离纯化α-CRY body，解析其分子结构组成，阐述应激环境下α-CRY body发生机理及生理功能。同时，本项目计划筛选出α-CRY body代谢调控相关的激活剂或抑制剂，为白内障的精准防治提供新的策略。

白内障是全球致盲率最高的眼科疾病，手术疗法仍是目前白内障治疗的唯一有效方案，暂无有效的预防和治疗药物。其发病组织是晶状体，晶状体内蛋白质受遗传或环境因素诱导会发生错误折叠，产生异常聚集变性而发生混浊是白内障产生的主要原因。在晶状体蛋白中，α-晶状体蛋白能够帮助细胞内其他蛋白质（例如β、γ晶状体蛋白）的正确折叠，防止发生错误折叠和聚集。为探究α-晶状体蛋白在维持蛋白质稳态中发挥的作用，我们通过体外纯化、免疫沉淀、流式分选等生物实验进行研究，发现体外纯化α-晶状体蛋白在紫外、过氧化氢等应激条件下，α-晶状体蛋白寡聚化，同时分子伴侣活性增强，可有效帮助突变蛋白BA3-G91Del正确折叠；α-晶状体蛋白在紫外、过氧化氢等应激条件下在胞内形成规则小体结构(α-CRY body)；该小体通过蛋白质间相互作用，在应激条件下快速凝集，以无膜细胞器的形式发挥功能，小体内富集错误折叠蛋白及泛素化相关蛋白，通过泛素化蛋白酶体降解途径降低错误折叠蛋白的聚集；药物筛选发现雷公藤红素靶向作用α-晶状体蛋白，并有效降低白内障突变蛋白的聚集。根据上述结果，我们认为α-晶状体蛋白在应激条件下通过小体结构发挥分子伴侣功能，帮助其他突变蛋白正确折叠，同时通过泛素化蛋白酶体途径降低错误折叠蛋白的聚集，并发现小分子药物雷公藤红素可激活该小体发挥功能，为白内障的精准防治提供了新的策略。