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X. Zhang, C. Marques, M. Gallagher, A. Taylor, P. Pereira, F. Shang; Glycation of Ubiquitin May Underlie the Molecular Mechanism of Diabetic Cataract . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2888.
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Purpose: The ubiquitin–proteasome pathway (UPP) plays critical roles in various cellular processes, such as protein quality control and stress responses. Glycation of cellular proteins by glucose or its metabolites may be a causal factor for many complications of diabetes. The objective of this study is to investigate the susceptibility of ubiquitin to glycation and to study the physiological consequences of glycation of ubiquitin in the lens or cultured human lens epithelial cells (HLEC). Methods: Ubiquitin was incubated with glucose–6–phosphate or methyglyoxal. The sites and extent of glycation were determined by mass spectrometry–based peptide mapping. The effects of glycated ubiquitin on proteolysis were determined using αA–crystallin as a substrate in reticlolyte lysate . The effects of methyglyoxal on the UPP were also determined in cultured HLEC. Results: Ubiquitin was readily glycated by glucose–6–phosphate and methyglyoxal. Whereas glucose–6–phosphate modifies ubiquitin primarily on Lys–11, methyglyoxal modifies ubiquitin mainly at Lys–48 and Arg–42. Both glucose–6–phosphate– and methyglyoxal–modified ubiquitin inhibit ATP–dependent degradation of αA–crystallin in reticulocyte lysates. Glycated ubiquitin was efficiently incorporated into poly–ubiquitin chains but the conjugates formed with glycated ubiquitin accumulated rather than being degraded by the 26S proteasome. Treatment of HLEC with methyglyoxal also resulted in accumulation of endogenous ubiquitin conjugates. Levels of ubiquitin conjugates in galactosemic cataract lenses were significant higher than those observed in control lenses. The accumulation of ubiquitin–conjugates was not due to inactivation of proteasome, because we found that the chymotrypsin–like peptidase activity of the proteasome was higher in methyglyoxal–treated HLEC or galactosemic cataract lenses. Conclusions: Ubiquitin is susceptible to glycation by glucose or its metabolites. Glycation of ubiquitin impairs functions of the UPP and results in severe physiological consequences. Modifications of ubiquitin by glucose or its metabolites may underlie the molecular mechanism of diabetic cataract.
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