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Abstract

Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, UV light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper (II)-induced aggregation. This work was motivated by the observation that zinc (II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper (II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper (II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.