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X. Zhang, A. Taylor, F. Shang; The Ubiquitin–Proteasome Pathway in the Lens Is a Target of Oxidative Stress . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3522.
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The ubiquitin–proteasome pathway (UPP) plays critical roles in many cellular processes, such as signal transduction, protein quality control and stress responses. Dysfucntion of the UPP is associated with several age–related degenerative diseases. The objective of this study is to investigate the susceptibility of the UPP to oxidative stress and its phyological consequences to lens cells.
Cultured human lens epithelial cells (HLEC) were exposed to constantly generated H2O2 for 4 h and proteasome activity was deteremined using fluorogenic peptides as substrates. Endogenous ubiquitin conjugates were determined by Western blotting and activities of ubiquitin conjuating enzymes were determined by thiolester assay. The modification of ubiquitin was determined by mass spectrometry–based peptide mapping. The effects of HNE–modified ubiquitin on proteolysis were determined using αA–crystallin as a substrate.
The proteasome in HLEC is suceptible to oxidative stress. Exposure to 40–50 µM H2O2 for 4 h resulted in 50–70% reduction in all the three peptidase activities of the proteasome. Although ubiquitin is relatively resistent to oxidation, it is readily modified by lipid peroxidation products, such as 4–hydroxynonenal (HNE). HNE–modified ubiquitin behaved as a dominant negative, which attenuates ubiquitination and inhibits ubiquitin–dependent degradation. Inhibition of proteasome activity in HLEC resulted in prolonged activation of the AKT pathway and diminished NF–ΚB activation. Inhibition of the proteasome pathway in HLEC also resulted in accumulation of oxidized proteins upon oxidative stress. Expression of mutant ubiquitin also rendered HLEC more susceptible to oxidative stress–inudced apoptosis.
The UPP is susceptible to oxidative inactivation. Impairment of the functions of the UPP disrupts signal transduction cascades and results in severe physiological consequences, incuding apoptosis of lens epithelial cells. In vivo, compromise of the UPP may be associated with cataract formation.
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