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Fu Shang, Zhenzhen Liu, Mingxing Wu, Xinyu Zhang, Yizhi Liu, Allen Taylor; Enhancement of Ubiquitination Promotes the Degradation of Mutant Crystallins in Lens. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6028.
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© ARVO (1962-2015); The Authors (2016-present)
The ubiquitin-proteasome pathway (UPP) is an important protein quality control mechanism which selectively degrades abnormal and damaged proteins. Ubiquitination is the first step of the UPP-mediated protein degradation. The objective of this work was to determine the effects of enhancement of ubiquitination capacity on intracellular degradation of cataract-causing mutant crystallins
Recombinant wild type (wt) and mutant γC-crystallins were expressed in bacteria and purified to homogeneity. The susceptibility of these crystallins to UPP-mediated degradation was assessed in lysates of human lens epithelial cells (HLEC). To investigate the effects enhancement of ubiquitination on accumulation and aggregation of the mutant crystallin, wt and mutant γC-crystallins were fused with green fluorescence protein (GFP) and co-expressed with a general ubiquitin conjugating enzyme Ubc5 and the bifunctional ubiquitin ligase/chaperone CHIP in HLEC. The intracellular aggregates of the GFP-γC-crystallin fusion proteins were monitored using fluorescence microscopy.
Wt γC-crystallin was resistant to degradation, but T5P-mutant γC-crystallin was rapidly degraded. Supplementation with Ubc5 or CHIP promoted the degradation of the mutant crystallin. When expressed in HLEC, wt γC-crystallin showed diffuse cytoplasmic distribution. However, the T5P- mutant γC-crystallin formed perinuclear aggregates. When CHIP and Ubc5 were co-expressed, the number and size of the aggregates of T5P- mutant γC-crystallin were significantly reduced.
The data indicate that the UPP selectively degrades mutant crystallins. Enhancement of ubiquitination capacity in lens cells reduces the aggregation of mutant crystallins. Together, these data also suggest that enhancement of UPP capacity is a valid strategy for prevention of accumulation and aggregation of abnormal proteins and cataract formation.
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