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T. Oya–Ito, B.–F. Liu, R.H. Nagaraj; Effect of Methylglyoxal Modification of Small Heat Shock Proteins on Caspases and Apoptosis in Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3607.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Small heat shock proteins (sHsps), alpha–crystallin and heat shock protein 27 (Hsp27) function as molecular chaperones and anti–apoptotic proteins. In a recent study, we demonstrated that both alpha–crystallin and Hsp27 become better chaperones after modification by low levels of methylglyoxal (MGO). In this study we have determined effects of MGO modification on their interaction with caspases, and staurosporine (SP)–induced apoptosis in lens epithelial cells. Methods: Native and MGO–modified proteins sHsps were introduced into human lens epithelial cells (HLE B–3) by encapsulation in a cationic lipid, BioPORTER. Apoptotic cells were assayed by annexin V staining. Effect on caspase 3, 7 and 9 activities and activation of caspase–3 were studied along with effect on the content of GSH and reactive oxygen species. Results: Introduction of alpha–crystallin and Hsp27 into lens epithelial cells inhibited apoptosis. MGO–modified proteins were more effective in inhibiting apoptosis than unmodified native proteins. In the presence of alpha–crystallin, caspase–3 like activity was reduced by 75% of that seen in cells treated with SP alone. MGO–modified alpha–crystallin did not affect caspase–3 activity. MGO–modified phosphorylated Hsp27 (pHsp27) markedly inhibited caspase–3 activity. MGO–modified alpha–crystallin almost completely blocked caspase–8 mediated activation of procaspase–3. Both MGO modified alpha–crystallin and Hsp27 reduced caspase–9 activity, and the effect was more pronounced with MGO–modified pHsp27. Both alpha–crystallin and Hsp27 blocked inactivation of caspase–7 and this property was unaffected by MGO–modification. In SP treated cells, ROS content was significantly reduced by alpha–crystallin and moderately reduced by Hsp27. While MGO–modification of alpha–crystallin did not change its ROS reducing capacity, it made Hsp27 a significantly better inhibitor of ROS formation. MGO–modification of alpha–crystallin did not change its inhibition of GSH loss in SP–treated cells. Conclusions: MGO–modified alpha–crystallin prevents procaspase–3 autocatalytic maturation mediated with caspase–8 and inhibits caspase–9 enzyme activity, while MGO–modified pHsp27 negatively regulates apoptosis by reduction of oxidative stress and by inhibition of caspase–3 and caspase–9 enzyme activities. Heat shock proteins may provide cells with better ability to cope with apoptotic stress after modification by MGO owing to multiple mechanisms of enhancement of chaperone function and inhibition of caspase activity.
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