Proteins in Molecular Mechanisms of Tear Film Formation

描述（由申请人提供）：本提案的长期目标是更好地了解泪液膜保护人类眼表的分子机制，并研究泪液中主要脂质结合蛋白泪液脂钙蛋白（TL）的蛋白-脂质相互作用。这些研究将有助于在对泪膜功能的科学理解的基础上开发干眼病的治疗方法。目的1：验证撕裂脂钙蛋白清除和溶解异常角膜表面脂质的假说。荧光标记脂质将被放置在人角膜上皮脱落。脂质运动将被追踪到整个泪液的覆盖溶液、耗尽泪液脂钙素的泪液和分离泪液成分的溶液中。成功将导致确定泪液脂钙素在干眼病（上皮侵蚀）中存在的异常角膜表面的保护作用。该实验是了解干眼病中影响泪膜稳定性和影响眼表的相互作用的关键。目的2：研究眼泪脂钙素在溶液中的脂肪酸结合位点。泪液脂钙蛋白中的配体结合位点将使用自旋标记的脂肪酸类似物来绘制，这种类似物可以猝灭顺序色氨酸突变体的荧光。成功将提供迫切需要的动态配体结合数据，包括配体在腔中的首选位置和三维运动的阐明。这些数据为探讨泪液脂钙素配体相互作用保护泪液膜的功能机制提供了结构基础。特异性目的3-进一步阐明pH诱导的泪液脂钙素花萼开放端环中发生的变化，从而调节配体结合。假设支架间环AB和GH有助于pH驱动机制影响配体结合，将使用荧光方法测试，以确定在酸性条件下环的附着。用pH滴定法监测主干运动、可及性和脂质结合的变化。这些实验将确定泪液脂钙素中调节配体结合的决定因素，并阐明所涉及的机制。干眼症是数百万美国人的困扰。这些研究将有助于了解人类泪膜的成分如何相互作用以保护健康的眼睛，并研究干眼异常表面上的这些相互作用，从而开发出有效的治疗方法。

DESCRIPTION (provided by applicant): 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.