The broad long-term objectives of this proposal are to understand better the molecular mechanisms by which the tear film protects the human ocular surface and to investigate the protein-lipid interactions of the principal lipid binding protein in tears, tear lipocalin (TL). The proposed studies will be useful in developing treatments for dry eye diseases based on a scientific understanding of tear film function.
Specific Aim 1 - To test the hypothesis that tear lipocalin scavenges and solubilizes lipids from the abnormal corneal surface. Fluorescent labeled lipids will be placed on human corneas with epithelial denudation. Lipid movement will be tracked into overlying solutions of whole tears, tears depleted of tear lipocalin and solutions of isolated tear components. Success will result in determination of the protective role of tear lipocalin on an abnormal corneal surface as that present in dry eye disease (epithelial erosions). The experiments are key to understanding of the interactions that influence tear film stability and affect the ocular surface in dry eye diseases.
Specific Aim 2 - To investigate the fatty acid binding site of tear lipocalin in solution. The ligand binding site in tear lipocalin will be mapped using spin labeled fatty acid analogs that quench the fluorescence of sequential tryptophan mutants. Success will provide critically needed dynamic ligand binding data including the preferred ligand positions in the cavity and elucidation of three dimensional (3D) motion. This data forms the structural basis to explore the functional mechanisms of tear lipocalin ligand interactions in protecting the tear film.
Specific Aim 3 - To further elucidate the pH induced changes that occur in the loops at the open end of the calyx of tear lipocalin that regulate ligand binding. The hypothesis that the interstand loops AB and GH contribute to a pH driven mechanism to influence ligand binding will be tested using fluorescent methods to determination apposition of the loops under acidic conditions. Changes in backbone motion, accessibility and lipid binding will be monitored with pH titration. These experiments will identify the determinants that regulate ligand binding in tear lipocalin and clarify the mechanisms involved. Dry eye is a problem for millions of Americans. The proposed studies will provide an understanding of how the components of the human tear film interact to protect the healthy eye and study these interactions on the abnormal surface of the dry eye, in order to develop effective treatments for this disorder. ? ? ? ?
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