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Fu Shang, Shasha Gao, Allen Taylor, Zhenzhen Liu, Yizhi Liu; Enhancement of Ubiquitin-Proteasome Pathway in Human Lens Epithelial Cells Reduces Aggregates of Mutant Crystallins. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4780.
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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. The objective of this work is to determine the effects of enhanced UPP capacity on intracellular accumulation and aggregation 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 of a specific E2 or E3 on 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. Addition of MG132, a proteasome inhibitor, blocked the degradation of T5P-mutant γC-crystallin, indicating that the degradation is UPP-mediated. When expressed in HLEC, wt γC-crystallin showed diffuse cytoplasmic distribution. However, the T5P- mutant γC-crystallin formed perinuclear aggregates. When UPP activity was enhanced by overexpression of Ubc5 or CHIP, the number and size of the aggregates of T5P- mutant γC-crystallin were significantly reduced. In contrast, inhibition of the proteasome increased the number of intracellular aggregates of of T5P- mutant γC-crystallin.
The data indicate that the UPP selectively degrades mutant crystallins. Enhancement of UPP activity 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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