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Glutathiolation Enhances the Degradation of C-crystallin in Lens and Reticulocyte Lysates, Partially via the Ubiquitin-Proteasome Pathway

¹From the Laboratory for Nutrition and Vision Research, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts; the ²Department of Ophthalmology, Institute of Clinical Neuroscience, and the ³Institute of Anatomy and Cell Biology, University of Göteborg, Sweden; and the ⁴Ophthalmic Research Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts.

PURPOSE. S-glutathiolated proteins are formed in the lens during aging and cataractogenesis. The objective of this work was to explore the role of the ubiquitin-proteasome pathway in eliminating S-glutathiolated C-crystallin.

METHODS. Recombinant human C-crystallin was mixed with various concentrations of glutathione (GSH) and diamide at 25°C for 1 hour. The extent of glutathiolation of the C-crystallin was determined by mass spectrometry. Native and S-glutathiolated C-crystallins were labeled with ¹²⁵I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. 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), were used to characterize the native and glutathiolated C-crystallins.

RESULTS. On average, two and five of the eight cysteines in C-crystallin were glutathiolated when molar ratios of C-crystallin-GSH-diamide were 1:2:5 and 1:10:25, respectively. Native C-crystallin was resistant to degradation in both lens fiber lysate and reticulocyte lysate. However, glutathiolated C-crystallin showed a significant increase in proteolytic degradation in both lens fiber and reticulocyte lysates. Proteolysis was stimulated by addition of adenosine triphosphate (ATP) and Ubc4 and was substantially inhibited by the proteasome inhibitor MG132 and a dominant negative form of ubiquitin, indicating that at least part of the proteolysis was mediated by the ubiquitin-proteasome pathway. Spectroscopic analyses of glutathiolated C-crystallin revealed conformational changes and partial unfolding, which may provide a signal for the ubiquitin-dependent degradation.

CONCLUSIONS. The present data demonstrate that oxidative modification by glutathiolation can render lens proteins more susceptible to degradation by the ubiquitin-proteasome pathway. Together with previous results, these data support the concept that the ubiquitin-proteasome pathway serves as a general protein quality-control mechanism.

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				X. Zhang, E. J. Dudek, B. Liu, L. Ding, A. F. Fernandes, J. J. Liang, J. Horwitz, A. Taylor, and F. Shang Degradation of C-terminal Truncated {alpha}A-crystallins by the Ubiquitin Proteasome Pathway Invest. Ophthalmol. Vis. Sci., September 1, 2007; 48(9): 4200 - 4208. [Abstract] [Full Text] [PDF]