泛素连接酶CHIP调控晶状体细胞蛋白质降解与再折叠的分子机制的研究

The accumulation and precipitation of damaged or misfolded proteins are causally related to cataract formation. There are presently no effective means to prevent the accumulation of damaged or misfolded proteins in the lens.The ubiquitin-proteasome pathway (UPP) and molecular chaperones are the two arms of the protein quality control system. The UPP plays an important role in selective removal of various forms of damaged lens proteins,while molecular chaperones are essential for refolding of unfolded and misfolded proteins. We and others previously demonstrated that componets of the UPP and chaperone system are present in both lens epithelial cells and lens fiber cells. The carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone with ubiquitin ligase activity, plays a pivotal role in coordinating the functions of the UPP and molecular chaperones in the protein quality control process. Our published data also showed that overexpression of CHIP in lens cells enhances degradation of mutant crystallins, preventing them from aggregating. Mechanisms of such intriguing phenomena remain to be elucidated. Based on these novel findings, we hypothesize that CHIP is a master regulator of the protein quality control process and that up-regulation of CHIP in lens cells prevents the accumulation of damaged proteins and cataractogenesis via promoting both removal and repair of damaged proteins. We are going to utilize a degradation-refolding bifunctional reporting protein, co-transfection experiments and a HSP70 promoter-luciferase reporting protein to thoroughly study roles of CHIP in protein degradation and refolding in lens epithelial cells. Then we are going to further validate these data in transgenic mice with CHIP overexpression specifically in the lens.Results of this study would provide a new target in treatment and prevention of cataract.

异常蛋白质在晶状体中的积累、聚集是白内障的主要成因。此前研究发现晶状体上皮和晶状体纤维细胞中均保留了泛素蛋白酶体通路与分子伴侣系统的主要组分，两者对降解或再折叠受损蛋白质，保持晶状体透明起着重要作用。我们前期研究发现泛素连接酶CHIP上调可减少晶状体上皮细胞内突变晶体蛋白形成的大分子量聚集物，且可增加部分分子伴侣的表达。故我们认为CHIP是晶状体细胞中联系上述两大系统的关键分子。本项目拟在晶状体上皮细胞中综合应用降解－再折叠双向报告蛋白、共转染实验和基于荧光素酶的带HSP70启动子的报告蛋白，系统研究CHIP增强蛋白质降解与再折叠的作用机制，进而利用晶状体特异性过表达CHIP的转基因小鼠获得CHIP对晶状体内异常蛋白质影响的直接证据。从清除异常蛋白质的两大策略降解和再折叠的"交汇点"入手，调控关键分子CHIP，为白内障防治提供新思路。

异常蛋白质在晶状体中积累、聚集是白内障的主要成因。课题组前期研究发现晶状体保留了泛素蛋白酶体通路(UPP)、分子伴侣系统的主要组分，这些组分对蛋白质质量控制起关键作用。我们已发现泛素连接酶CHIP上调可减少晶状体上皮细胞内突变晶体蛋白形成的大分子量聚集物、提高晶体蛋白水溶性，提示CHIP是晶状体细胞中联系UPP与分子伴侣的关键分子。.本项目分别通过上调、下调晶状体上皮细胞中CHIP含量，系统研究CHIP在晶状体细胞蛋白质质量控制中的作用。我们发现：1.晶状体上皮细胞经慢病毒转染针对CHIP的ShRNA，CHIP表达下调。热休克处理后，CHIP下调组蛋白质泛素化水平降低，异常聚集物增多；对照组蛋白质泛素化水平升高，分子伴侣Hsp27、Hsp70表达量增加。2. 晶状体上皮细胞转染编码CHIP的质粒后，CHIP表达上调，蛋白质泛素化水平升高，异常晶体蛋白聚集物减少。综合上述结果，CHIP同时通过UPP与分子伴侣起作用，增强晶状体细胞内异常蛋白质的清除和再折叠，有利于保持晶状体透明性，可能是防治白内障的有效分子靶点。.项目资助发表SCI论文4篇，待发表1篇。以本课题为主要研究方向的硕士生1名，已获硕士学位。项目投入经费23万元，支出15.98万元，各项支出基本与预算相符。剩余经费7.02万元，剩余经费计划用于本项目研究后续支出。