Project Summary: Our laboratories have focused on the most prevalent chemical modifications that we have identified to be associated with the insoluble proteins present in the nucleus of the lens- deamidation and oxidation. These modifications are most relevant to age-related nuclear cataract, by far the most common type of cataract. In this proposed work, we will examine the interplay between deamidation and oxidation in order to mimic the age-related processes in the lens. Although the lens environment is normally in a reduced state, oxidation of sulfhydryls in crystallins has long been associated with age-related cataract as the pool of lens glutathione diminishes with aging in the center of the lens. The formation of non-native, disulfide crosslinked crystallin subunits via the oxidation of Cys residues is therefore anticipated to be a key process leading to the aggregation and insolubilization of lens proteins. In Aim 1, we will determine how specific, age-related deamidations in γS promote its aggregation by identifying non-native disulfide bond formed in response to combined deamidation and oxidation. In Aim 2, we will test the hypothesis that non-native disulfide bond formation leads to higher ordered oligomers of γS. While the focus of Aims 1 and 2 is the oxidation of deamidated γS, Aim 3 explores whether these age-related modifications in γS lead to non-native crosslinks between γ- and β-crystallin subunits and thereby disrupt the native quaternary arrangement of the crystallins. Overall, these findings will elucidate how deamidation, a spontaneous modification, contributes to the oxidation-driven aggregation cascade that underlies age-related nuclear cataract. Establishing that deamidation mediates its effects predominantly via augmenting the oxidation of crystallin proteins will provide a robust model for the development of therapeutic strategies aiming to delay the onset of age-related nuclear cataract by restoring the antioxidant levels of the lens. The work will also have significant implications for several other diseases where deamidation and oxidation of long-lived proteins is associated with amyloid fibril formation.