Dual Functions of Ubiquitin–Proteasome Pathway in Lens Protein Quality Control

Abstract

Abstract: : Purpose: Accumulation of damaged or denatured proteins is associated with cataract. The purpose of this study is to determine roles of ubiquitin–proteasome pathway (UPP) in removal and repair of damaged proteins. Methods:Cutured human lens epithelial cells (HLEC) were exposed to physiologically relevant level of H₂O₂ (20 µM) chronically generated by the glucose/glucose oxidase system. Levels of protein carbonyls were determined as a marcker of oxidized proteins. To determine the chaperone–like activity of the UPP, heat–denatued luciferase was used as a model substrate and the UPP was reconstituted using purified components. Chaperone–like activity of the proteasome was determined by its ability to restore the activity of heat–denatured luciferase. Results: Exposure of HLEC to 20 µM H₂O₂ alone for 8 hours resulted in a marginal increase in levels of oxidized proteins. However, if the proteasome activity was inhibited, this level of H₂O₂ resulted in a 3–fold increase in levels of oxidized proteins. Inhibition of the proteasome also enhanced the cytotoxicity of H₂O₂ to HLEC. Futhermore, the UPP exibits a chaperone–like activity which can refold denatured proteins. Denatured proteins were preferentially ubiquitinated by the ubiquitin conjugation enzymes. The ubiquitinated proteins were targeted to the 26S proteasome for renaturation or degradation. We further demonstrated that the UPP employs the same mechanism as heat shock proteins to recognize damaged proteins and that the UPP competes with other cellular chaperones for proteins with abnormal structures. Conclusions: These data demonstrate that the UPP is an important protein quality control machinery in the lens, which selectively recognizes proteins with abnormal structures. The dual functions of the UPP in protein quality control assure the efficient removal or repair of damaged proteins. Dysfunction of this protein quality control mechanism may result in accumulation of damaged proteins in the cells and may be involved in the pathophysiology of cataract.

Keywords: proteolysis • chaperones • oxidation/oxidative or free radical damage