Enhancement of ubiquitin conjugation activity reduces intracellular aggregation of V76D mutant γD-crystallin

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1.

V76D-γD-crystallin forms aggregates in the nucleus and cytosol of transfected cells. Representative fluorescence microscopy images of human lens epithelial cells SRA01/04 and HeLa cells transfected with RFP-wt-γD-crystallin (A) and RFP-V76D-γD-crystallin (B). Whereas wt-γD-crystallin was evenly distributed throughout the cells, V76D mutant γD-crystallin formed aggregates in the nucleus and cytosol.

Figure 2.

Cytosol aggregates of V76D-γD-crystallins accumulate in the ER in transfected cells. Confocal sectioned images of human lens epithelial cells SRA01/04 and HeLa cells transfected with RFP-V76D-γD-crystallin and GFP-ER-targeting peptide. V76D-γD-crystallins form aggregates in nucleus and cytoplasm. Most of the cytosol aggregates are found to be localized in the ER.

Figure 3.

Decreased solubility of V76D-γD-crystallin. HeLa cells were transfected with either RFP-wt or RFP-V76D mutant γD-crystallins. The insoluble and soluble fractions were separated and resolved by SDS-PAGE and probed using anti-RFP (A). The bands identified by the antibody were then quantified with ImageJ (B). Eight percent of V76D-γD-crystallin was found in the soluble fraction compared to 80% of wild type, indicating a dramatic decrease in the solubility due to the point mutation.

Figure 4.

Quantification of the proportion of transfected cells with V76D-γD-crystallin aggregates. The percentage of human lens epithelial cells (A) or HeLa cells (B) with V76D aggregates when cotransfected with empty vector (as control), CHIP, or Ubc5, respectively, was quantified. Overexpression of CHIP or Ubc5 significantly reduced percentage of cells with V76D-γD-crystallin aggregates (P < 0.05 when compared to control without MG132). Proteasome inhibitor MG132 attenuated the effects of CHIP and Ubc5 on reducing V76D aggregates. For each experiment, five low-magnification views, each containing approximately 300 cells, were picked randomly from each group, and the number of cells with aggregates was counted. Each bar represents an average of three independent experiments.

Figure 5.

CHIP and Ubc5 diminish V76D-γD-crystallin aggregates in human lens epithelial cells. Diminution of aggregates involves UPP-dependent proteolytic activity. Human lens epithelial cells were transfected with RFP-V76D-γD-crystallin and EGFP (as control) or EGFP-CHIP or EGFP-Ubc5. (A) Coexpression of CHIP and Ubc5 reduced the percentage of transfected cells with V76D-γD-crystallin aggregates. (B) Proteasome inhibitor MG132 attenuated the effects of CHIP and Ubc5, increasing the percentage of cells with aggregates. (C) Distribution of ratio of RFP/GFP peak intensity in individual cell.

Figure 6.

Overexpression of CHIP or Ubc5 reduced the amount of V76D mutant γD-crystallin. HeLa cells were cotransfected with V76D mutant γD-crystallin and either vector alone, CHIP, or Ubc5. The soluble (A) and insoluble (C) fractions were separated and resolved by SDS-PAGE and probed using anti-RFP. The bands identified by the antibody were then quantified with ImageJ (B, D). Overexpression of both CHIP and Ubc5 reduced V76D-γD-crystallin in the insoluble fractions (panel D, left side, P < 0.05). Proteasome inhibitor, MG132, attenuated the effects of CHIP or Ubc5, resulting in greater accumulation of V76D-γD-crystallin in both soluble and insoluble fractions.

Figure 7.

Overexpression of CHIP and Ubc5 did not significantly reduce the amount of wild type γD-crystallin. HeLa cells were cotransfected with wt-γD-crystallin and either vector alone, CHIP, or Ubc5. The soluble (A) and insoluble (C) fractions were separated and resolved by SDS-PAGE and probed using anti-RFP. The bands identified by the antibody were then quantified with ImageJ (B, D). Overexpression of CHIP did not affect the amount of wt-γD-crystallin; overexpression of Ubc5 reduced the level of wt-γD-crystallin, but not in a statistically significant manner.

Figure 8.

The effects of CHIP require functional TPR and the U-box domains. Cells were transfected with RFP-V76D-γD-crystallin and vector alone, wild type His-tagged-CHIP, c-myc-ΔTPR-CHIP, or c-myc-ΔU-box-CHIP, respectively. (A) Nuclei were stained with DAPI, and the aggregation of V76D in each group was compared with fluorescence microscopy. The soluble (B) and insoluble (C) fractions were separated by SDS-PAGE and probed using anti-RFP antibody to compare the level of V76D-γD-crystallins.