The long term goal of the proposed project is a better understanding of the multiple roles of alpha-crystallin in the lens fiber cells. Alpha- crystallin has long been thought to be involved with lens transparency maintenance, thus knowledge of its physical properties will likely lead to increased understanding of cataract onset and may even lead to the development of improved cataract treatments.
The specific aims of this proposal include the quantitative analysis of the chaperone-like activity of alpha-crystallin and its subunits, elucidation of the binding site(s) responsible for chaperone-like activity, the localization of the non-chaperone associated binding site(s), and the identification of lens-specific factors that bind to alpha-crystallin. The chaperone-like activity will be analyzed using a quantified insulin reduction assay which is based on alpha- crystallin's ability to suppress reduced insulin B chain aggregation. The kinetic differences between alphaA crystallin and alphaB crystallin will be studied using this assay. Chaperone associated binding site(s) will be identified with the use of a radiolabeled crosslinking agent, [125I] PEAS, and peptide sequencing. The crosslinking will be confirmed through site-directed mutagenesis of the implicated domains in alpha- crystallin. Other alpha-crystallin interactions will be accessed using immobilized lens cytoskeletal complexes and alphaA crystallin in binding studies that will include chimeric versions of alphaA crystallin and lens cell homogenates. The chimeric proteins will be used specifically to localize the binding site(s) responsible for these non-chaperone associated interactions.
