Sulfur-centered post-transnational modifications of the crystallin are a recurring feature in many cataractous lenses. This is especially true of maturity-onset human nuclear cataract, in which the cysteine and methionine residues of the Beta and gamma crystallin are oxidized. However, no direct link has been established between such protein modifications, and the formation of lens opacities. In this proposal, a strategy is presented to establish such a link in vitro, between modifications of the cysteine and methionine residues of the gamma crystallin and the two molecular mechanisms that lead to opacity: protein aggregation and protein phase separation. Our hypothesis is that - not all sulfur-centered modifications are cataractogenic, and that molecular factors such as charge and hydrophilicity are essential elements that determine the role of a particular modification in opacification. To test this hypothesis, the following specific aims are proposed: (1) Introduce in vitro, oxidative modifications normally found in the lens at the sulfur centers of the gamma crystallin, and measure the effect of these modifications on the aggregation and phase separation properties of the protein solutions. (2) Determine the role played by hydrophilicity in sulfur-centered modifications, on lens proteins aggregation and phase separation. (3) Evaluate the role of a crystallin in inhibiting protein aggregation, due to sulfur-centered modifications of the gamma crystallin. (4) Determine the role of individual cysteine and methionine residues in oxidative modifications by introducing point mutations at these residues, using site-directed mutagenesis. These studies are expected to provide the basis for the identification of potentially cataractogenic crystallin modifications in vivo. Since the sulfur centers can be viewed also as anchors for modifying groups, our conclusions should be broadly applicable to all post-transnational protein modifications found in the lens.
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