State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Invest Ophthalmol Vis Sci. 2012 Sep 25;53(10):6655-65. doi: 10.1167/iovs.12-9744.
The ubiquitin-proteasome pathway (UPP) is an important protein quality control mechanism for selective degradation of abnormal proteins. The objective of this study was to test the hypothesis that enhancement of the UPP capacity could attenuate the accumulation and aggregation of misfolded proteins using V76D-γD-crystallin as a model substrate.
Wild type (wt) and V76D mutant γD-crystallin were fused to red fluorescence protein (RFP) and expressed in human lens epithelial cells. The cellular distribution of the expressed proteins was compared by fluorescence microscopy. The solubility of wt- and V76D-γD-crystallin was determined by cellular fractionation and Western blotting. Wt-γD-RFP and V76D-γD-RFP were also cotransfected along with a ubiquitin ligase (CHIP) or a ubiquitin-conjugating enzyme (Ubc5) into cells. Levels of wt- and V76D-γD-crystallin, the percentage of transfected cells with aggregates, and aggregate size were quantified and compared among different groups.
Wt-γD-crystallin was evenly distributed in cells, whereas V76D-γD-crystallin formed intracellular aggregates. Eighty percent of wt-γD-crystallin was detected in the soluble fraction, whereas only 7% of V76D-γD-crystallin was soluble. CHIP or Ubc5 coexpression reduced the protein level of V76D-γD and concomitantly its aggregation in transfected cells; these effects could be attenuated by proteasome inhibitor. Mutant CHIP with defect TPR (tetratricopeptide repeat) or U-box domain failed to reduce levels of V76D-γD-crystallin.
Enhancing ubiquitin conjugation activity reduces accumulation and aggregation of V76D-γD-crystallin by promoting its degradation. Upregulation of ubiquitin-conjugating activity could be an effective strategy to maintain lens transparency by eliminating other forms of misfolded proteins.
泛素-蛋白酶体途径（UPP）是一种重要的蛋白质质量控制机制，可选择性降解异常蛋白质。本研究旨在通过以 V76D-γD-晶体蛋白作为模型底物来检验以下假设：增强 UPP 能力可以减少错误折叠蛋白质的积累和聚集。
将野生型（wt）和 V76D 突变γD-晶体蛋白与红色荧光蛋白（RFP）融合，并在人晶状体上皮细胞中表达。通过荧光显微镜比较表达蛋白的细胞分布。通过细胞分级分离和 Western 印迹法测定 wt-和 V76D-γD-晶体蛋白的溶解度。还将 wt-γD-RFP 和 V76D-γD-RFP 与泛素连接酶（CHIP）或泛素结合酶（Ubc5）共转染到细胞中。定量比较不同组之间 wt-和 V76D-γD-晶体蛋白的水平、具有聚集体的转染细胞的百分比以及聚集体的大小。
wt-γD-晶体蛋白均匀分布在细胞中，而 V76D-γD-晶体蛋白形成细胞内聚集体。80%的 wt-γD-晶体蛋白检测到在可溶性部分，而 V76D-γD-晶体蛋白仅 7%可溶。CHIP 或 Ubc5 共表达降低了转染细胞中 V76D-γD 的蛋白水平及其聚集；这些作用可以通过蛋白酶体抑制剂减弱。具有缺陷 TPR（四肽重复）或 U 盒结构域的突变 CHIP 不能降低 V76D-γD-晶体蛋白的水平。
增强泛素缀合活性通过促进其降解来减少 V76D-γD-晶体蛋白的积累和聚集。上调泛素结合活性可能是通过消除其他形式的错误折叠蛋白质来维持晶状体透明性的有效策略。
