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E.J. Dudek, A. Taylor, X. Zhang, B. Liu, J.J. Liang, F. Shang; Glutathiolation of gC–Crystallin Reveals a Degradation Signal for the Ubiquitin Proteasome Pathway . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2018.
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© ARVO (1962-2015); The Authors (2016-present)
To determine how glutathiolation renders lens proteins more susceptible to ubiquitin–dependent proteolysis.
Full length γC–crystallin was expressed in E coli and purified to homogeneity. Glutathiolation was performed by incubation with GSH in the presence of diamide. Deglutathiolation of γ–crystallin was accomplished using incubation with DTT. Conformational changes were monitored by far UV circular dichroism, tryptophan fluorescence intensity and binding to the hydrophobic fluorescence probe 4,4’–dianilino–1,1’–binaphthalene–5,5’–disulfonic acid (Bis–ANS). Degradation was performed in reticulocyte lysate using 125I–labelled γC–crystallins as substrates.
Glutathiolation of γC–crystallin resulted in significant conformational changes as indicated by decreased tryptophan fluorescence and increased binding to Bis–ANS and altered far UV circular dichroism spectra. Whereas unmodified γC–crystallin is resistant to proteolysis, glutathiolated γC–crystallin was susceptible to ATP–dependent degradation. About 8% of glutathiolated γC–crystallin degraded in a 90 min degradation assay. Addition of Ubc4 enhanced the degradation and depletion of ubiquitin–activating enzyme diminished the degradation, indicating that glutahiolated γC–crystallin is degraded by the ubiquitin–proteasome pathway. Although degluatiolation only partially restored the conformation, it totally abolished the susceptibility to ubiquitin–dependent degradation. Alkylation with iodoacetamide also rendered gC–crystallin more susceptible to ubiquitin–dependent degradation, although it only caused marginal conformational changes. This phenomenon is not limited to γC–crystallin. Glutathiolation of carbonic anhydrase also triggered its degradation by the ubiquitin–proteasome pathway.
Glutathiolation or other types of modification of thiol groups of lens proteins, possibly along with structural changes, render them better substrates for ubiquitin–dependent proteolysis. These data suggests that glutathiolation and other types of thiol modification may serves as signal for ubiquitin–dependent degradation. Since glutathiolation increases upon oxidative stress, these data further indicate that the ubiquitin–proteasome pathway is an important protein quality control mechanism, which selectively degrades redox–modified proteins.
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