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M. Zetterberg, A. Taylor, J.N. Liang, F. Shang; GSH–Modified GammaC–Crystallin is Selectively Degraded by the Ubiquitin–Proteasome Pathway . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3891.
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Purpose: To explore the ubiquitin–proteasome pathway (UPP) as an important protein quality control process by which cells selectively degrade and remove damaged proteins. γ–crystallin is susceptible to formation of mixed disulfides with GSH upon oxidative stress. The aim of this study was to investigate if the UPP also degrades γ–crystallin modified by GSH. Methods: In order to produce GSH–modified γC–crystallin, recombinant human γC–crystallin was mixed with GSH and diamide at molar ratios of 1:10:5 (γC–crystallin:GSH:diamide). After incubation at 23°C for 4 hours the remaining GSH and diamide were removed by gel filtration. The extent of modification, i.e. glutathiolation of the γC–crystallin, was determined by mass spectrometry. Normal and GSH–modified γC–crystallins were labelled with 125 I and subsequently incubated with whole reticulocyte lysate at 37°C for 90 min to determine ATP–dependent and ATP–independent proteolysis, using the reticulocyte lysate as a source of ubiquitinating and proteolytic capability. Parallel experiments included the proteasome inhibitor MG132 (40 µM). Results: Five of the 8 cysteines in γC–crystallin were readily modified by GSH to form mixed disulfide. Unmodified γC–crystallin was resistent to degradation by reticulocyte lysate. In contrast, GSH–modified γC–crystallin showed 8% (ATP–independent) and 12% (ATP–dependent) proteolytic degradation respectively. A majority of the ATP–dependent proteolysis was inhibited by 40 µM MG132, suggesting that the UPP was responsible for this degradation. Conclusions: The present data demonstrate that formation of mixed GSH–containing disulfides can render proteins more susceptible to degradation by the UPP, supporting a role for this proteolytic pathway in degradation of oxidatively damaged proteins. Failure of this process may be etiologically related to cataract.
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