Abstract
Purpose:
Accumulation and precipitation of abnormal proteins in the lens is causally related to cataract formation. The ubiquitin proteasome system (UPS) is an important mechanism for identification and selective removal of abnormal proteins. Age-or stress-related compromise to the UPS contributes to accumulation of abnormal proteins in the lens. The purpose of this study is to test whether overexpression of CHIP, a bifunctional molecule that participates in ubiquitin conjugation and chaperone function, or Ubc5, a ubiquitin conjugating enzyme with broad substrate specificity, or CHIP and Ubc5 together increase ubiquitination and UPS-dependent degradation of abnormal proteins from lens cells.
Methods:
Overexpression of CHIP and Ubc5 in cultured human lens epithelial cells (HLEC) was achieved using adenovirus-mediated transfection. Levels of CHIP, Ubc5 and ubiquitin conjugates were determined by Western blotting. The capacity to catalyze de novo ubiquitination was determined using 125I-labeled ubiquitin and endogenous substrates or using 125I-labeled luciferase as a specific substrate, with supplemental ubiquitin. UPS-dependent degradation capacity was determined using 125I-labeled luciferase.
Results:
Adenovirus-mediated overexpression increased the levels of CHIP and Ubc5 by 2-5 fold relative to control cells. When both CHIP and Ubc5 were overexpressed in the cells, a majority of the overexpressed CHIP was in mono-ubiquitinated form. Overexpression of CHIP alone did not effect ubiquitination or ubiquitin-dependent degradation. Overexpression of Ubc5 increased ubiquitination and UPS-dependent degradation by ~20%. However, when both CHIP and Ubc5 were overexpressed, ubiquitination and UPS-dependent degradation increased by >50%.
Conclusions:
Levels of CHIP and Ubc5 HLEC are limited in lens cells. Overexpression of CHIP and Ubc5 together increases the ubiquitination and UPS-dependent degradation capacity. These data suggest that ubiquitination is the rate-limiting step of UPS-mediated degradation and that enhancement of UPS capacity can accelerate the degradation of abnormal proteins. These studies open up new avenues for delaying cataract.
Keywords: 445 cataract •
413 aging •
662 proteolysis