Lens cells have a ubiquitin–dependent chaperone activity

Abstract

Abstract: : Purpose: Accumulation of damaged or denatured proteins is associated with cataract. Efficient removal of damaged proteins or renaturation of the denatured proteins is crucial for the maintenance of lens transparency. We previously demonstrated that ubiquitin is involved in removal of oxidized proteins. The objective of this project is to determine if ubiquitin has a role in renaturation of denatured proteins. Methods: To determine the role of ubiquitin in facilitating protein refolding and solubilization in a cell–free system, lens proteins were extracted from the outer cortex of calf lenses, labeled with ¹²⁵I, and incubated with a ubiquitin–depleted fraction from red blood cells at 38 ^oC for 60 min, with or without additional ubiquitin. Then the radioactivities in the soluble and insoluble were determined. To test the chaperone activity in lens cells, firefly luciferase was first heat–denatured and then delivered into cultured lens epithelial cells in the presence or absence of K6W mutant ubiquitin which allows for ubiquitination, but not degradation or deubiquitination. Luciferase activity in the cells was determined to monitor the chaperone activity. Results: When ubiquitin was depleted, ∼39% of ¹²⁵I–labeled lens proteins precipitated during the incubation at 38 ^oC. However, if ubiquitin was added to the incubation mixture, only 18% of the lens proteins precipitated during the same period of incubation. When delivered into lens epithelial cells, 20–30% of the heat–denatured luciferase was reactivated. Overexpression of the K6W mutant ubiquitin stabilized denatured luciferase, but it significantly inhibited the renaturation of denatured luciferese. Consistent with the stabilization of denatured luciferase, higher levels of ubiquitinated luciferase were detected when K6W ubiquitin was expressed in the cells. Conclusion: These results demonstrated for the first time that ubiquitin is involved in renaturation of denatured proteins. Together with its role in targeting damaged proteins for degradation, this work indicates that the ubiquitin–proteasome pathway is a master cellular protein quality control mechanism.

Keywords: chaperones • protein structure/function • cataract