Abstract
Purpose: :
Previously we have shown that enhanced ubiquitin conjugating activity could reduce intracellular aggregates of cataract-causing mutant crystallins. The objective of this study was to investigate the mechanisms by which enhancements of ubiquitin conjugating activity reduced the aggregates of mutant crystallins in the cells.
Methods: :
Two cataract-associated mutant crystallins, T5P- γC-crystallin and V76D- γD-crystallin as well as their corresponding wild type, were fused with red fluorescence protein (RFP) and expressed in human lens epithelial cells (HLEC) and HeLa cells. To investigate the selectivity of a ubiquitin ligase CHIP, and a ubiquitin conjugate enzyme Ubc5, in degradation of mutant crystallins, wt and mutant γ- crystallins were co-transfected with either CHIP or Ubc5 into HLEC or HeLa cells. The intracellular aggregates of the RFP- γ- crystallin fusion proteins were quantified using fluorescence microscopy. The protein levels of both wild type and mutant crystallins were determined with Western blotting analysis. Cycloheximide chase assay was conducted to compare the degradation rates of wt versus mutant crystallin in the presence of CHIP or Ubc5. To determine ubiquitination of the crystallins in the cells, wt- and mutant γC-crystallin were co-transfected with His-tagged K6W-ubiquitin. The ubiquitinated RFP- γ- crystallins were isolated with Ni-NTA column, and were identified by Western blotting analysis.
Results: :
Wild type γC- and γD-crystallins were evenly distributed in cytoplasm, whereas T5P- γC- and V76D- γD- crystallins formed perinuclear and nuclear aggregates, respectively. Over-expression of either CHIP or Ubc5 reduced the number and size of aggregates of the mutant T5P-γC- and V76D- γD- mutant γ-crystallins. Overexpression of Ubc5 generally enhanced the degradation of both wild type and mutant-crystallins. However, expression of CHIP selectively enhanced the degradation of mutant γ-crystallins. This result was further confirmed by cycloheximide chase assay. Consistent with the selective degradation of mutant γ- crystallins, only mutant γ-crystallins were found ubiqutinated in the cells.
Conclusions: :
The data show that both CHIP and Ubc5 could reduce aggregation of mutant crystallins by promoting their degradation. However, it appears that CHIP has greater selectivity than Ubc5 in promoting the degradation of mutant crystallins. Together, these data suggest that enhancement of CHIP activity in the lens may be a valid strategy for selective removal of mutant and abnormal proteins in order to prevent their accumulation and aggregation in lens.
Keywords: proteolysis • proteins encoded by disease genes • protective mechanisms