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

Abstract

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 gammaC-crystallin. Recombinant human gammaC-crystallin was mixed with various concentrations of glutathione (GSH) and diamide at 25 degrees C for 1 hour. The extent of glutathiolation of the gammaC-crystallin was determined by mass spectrometry. Native and S-glutathiolated gammaC-crystallins were labeled with (125)I, 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 gammaC-crystallins. On average, two and five of the eight cysteines in gammaC-crystallin were glutathiolated when molar ratios of gammaC-crystallin-GSH-diamide were 1:2:5 and 1:10:25, respectively. Native gammaC-crystallin was resistant to degradation in both lens fiber lysate and reticulocyte lysate. However, glutathiolated gammaC-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 gammaC-crystallin revealed conformational changes and partial unfolding, which may provide a signal for the ubiquitin-dependent degradation. 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.