Proteins in Molecular Mechanisms of Tear Film Formation

泪膜形成分子机制中的蛋白质

基本信息

批准号：
7454260
负责人：
BEN J GLASGOW
金额：
$ 36.75万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-02-01 至 2011-06-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：该提案的广泛长期目标是更好地了解泪膜保护人眼表面的分子机制，并研究泪液中主要脂质结合蛋白的蛋白质-脂质相互作用，泪液脂质运载蛋白（TL）。拟议的研究将有助于基于对泪膜功能的科学理解开发干眼病的治疗方法。具体目标 1 - 检验泪液脂质运载蛋白清除并溶解异常角膜表面脂质的假设。荧光标记的脂质将被放置在上皮剥脱的人角膜上。脂质运动将被追踪到全泪液、泪液脂质运载蛋白耗尽的泪液和分离的泪液成分的溶液的覆盖溶液中。成功将导致确定泪液脂质运载蛋白对干眼病（上皮糜烂）中存在的异常角膜表面的保护作用。这些实验是了解干眼病中影响泪膜稳定性和眼表相互作用的关键。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。将使用自旋标记的脂肪酸类似物来绘制泪液脂质运载蛋白中的配体结合位点，该类似物可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，包括腔内的首选配体位置和三维 (3D) 运动的阐明。该数据构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。具体目标 3 - 进一步阐明调节配体结合的泪液脂质运载蛋白花萼开口端环中发生的 pH 诱导变化。跨位环AB和GH有助于影响配体结合的pH驱动机制的假设将使用荧光方法进行测试，以确定酸性条件下环的并置。骨干运动、可及性和脂质结合的变化将通过 pH 滴定来监测。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面临的一个问题。拟议的研究将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的这些相互作用，以便开发针对这种疾病的有效治疗方法。