Human γS-Crystallin–Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage

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, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking 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 γScrystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in 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.

Chemical Properties Determine Solubility and Stability in βγ‐Crystallins of the Eye Lens

“…Recent evidence suggests that disulfide bond formation between solvent exposed cysteines in γ-crystallins may serve as a buffering system, through disulfide exchange, to prevent harmful oxidations. [57,221,246] Formation of these dimers appears to be a last resort buffer, as high concentrations of these dimers eventually promote the formation of larger aggregates. The mechanisms by which aggregates are formed in the eye lens, whether they be amorphous-looking aggregates, amyloid fibrils, or a combination of the two, is still largely unknown.…”

“…[53] Amorphous-looking aggregates have been observed for HγD in the presence of copper and zinc ions [54] and for the HγD-P23T variant, [55] whereas HγS and its G18V variant display a mixture of amorphous aggregates and amyloid fibrils, with the relative populations varying based on the aggregation conditions. [56,57] Amyloid fibrils have also been observed in UV-induced cataracts in both porcine [58] and human lenses, [59] raising the question of how prevalent each type of aggregate is, and under what conditions. A characteristic signature has been identified in the 2DIR spectrum that can detect amyloid secondary structure, even if the individual domains contain only 4-5 β-strands.…”

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

“…Together, these four residues have been proposed to form a cysteine tetrad that shuffles intramolecular disulfides to maintain γS in a soluble, monomeric form. 49 Our findings suggest that deamidation-increased dynamics of γS disrupt such intramolecular disulfides and thereby increase the susceptibility of γS to non-native intermolecular crosslinks, such as those observed between C24 residues. 39,59 Future studies are needed to determine if other cysteine residues are modified and are therefore associated with the aggregation of the different deamidated γS-crystallins.…”

“…In the solution structure of γS, C82 is in close proximity to C22 and C26 (Figure 9e) which are, in turn, proximal to the solvent‐exposed C24. Together, these four residues have been proposed to form a cysteine tetrad that shuffles intramolecular disulfides to maintain γS in a soluble, monomeric form 49 . Our findings suggest that deamidation‐increased dynamics of γS disrupt such intramolecular disulfides and thereby increase the susceptibility of γS to non‐native intermolecular crosslinks, such as those observed between C24 residues 39,59 .…”

“…Three cysteines, C22, C24, and C26, form part of a distinctive sequence, 21-DCDCDC-26, that has been proposed to act as a redox center and involve the formation of at least one intramolecular disulfide bond, 47,48 most likely between C22 and C26, as occurs in the γS disulfidelinked dimer. 39 In view of a recent report indicating disulfide interchange at this site, 49 we investigated whether increased dynamics in this region, based on our recent NMR data, 37 would disrupt the disulfide arrangement. After buffer exchange to lower reducing agent and incubation under native conditions for 60 min at 37 C, mass peaks of the γS 20-35 tryptic peptide containing an internal disulfide crosslink and one alkylation were 3-4 times higher in WT than in TM (arrows in Figure 9a,b, respectively).…”