Proteins in Molecular Mechanisms of Tear Film Formation

描述（由申请人提供）：本提案的广泛长期目标是更好地了解泪膜保护人眼表的分子机制，并研究泪液中主要脂质结合蛋白泪液脂质运载蛋白（TL）的蛋白质-脂质相互作用。基于对泪膜功能的科学理解，拟议的研究将有助于开发干眼病的治疗方法。具体目的1-检验泪液脂质运载蛋白从异常角膜表面清除和溶解脂质的假设。将荧光标记脂质置于上皮剥脱的人角膜上。脂质运动将被追踪到整个泪液的覆盖溶液、泪液脂质运载蛋白耗尽的泪液和分离的泪液组分的溶液中。成功将导致确定泪液脂质运载蛋白对异常角膜表面的保护作用，如干眼病（上皮糜烂）中存在的保护作用。这些实验是理解干眼症中影响泪膜稳定性和眼表的相互作用的关键。具体目标2-研究溶液中泪液脂质运载蛋白的脂肪酸结合位点。泪液脂质运载蛋白中的配体结合位点将使用自旋标记的脂肪酸类似物进行映射，所述脂肪酸类似物猝灭连续色氨酸突变体的荧光。成功将提供急需的动态配体结合数据，包括腔中的优选配体位置和三维（3D）运动的说明。这些数据形成了探索泪液脂质运载蛋白配体相互作用在保护泪膜中的功能机制的结构基础。具体目的3-为了进一步阐明pH诱导的变化，发生在环的开放端的泪脂质运载蛋白的萼，调节配体结合。将使用荧光方法检测支架间环AB和GH促成pH驱动机制以影响配体结合的假设，以确定酸性条件下环的并置。将采用pH滴定法监测骨架运动、可及性和脂质结合的变化。这些实验将确定调节泪液脂质运载蛋白中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问题。拟议的研究将提供对人类泪膜成分如何相互作用以保护健康眼睛的理解，并研究干眼症异常表面上的这些相互作用，以便开发有效的治疗方法。

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.