Proteins in Molecular Mechanisms of Tear Film Formation

描述(申请人提供)：这项建议的广泛的长期目标是更好地了解泪膜保护人类眼表的分子机制，并研究泪液中主要的脂质结合蛋白-泪液脂结合蛋白(TL)的蛋白质-脂相互作用。建议的研究将有助于基于对泪膜功能的科学理解来开发干眼症的治疗方法。具体目标1-验证泪液Lipocalin清除和溶解异常角膜表面的脂类的假设。荧光标记的脂质将被放置在有上皮剥离的人的角膜上。脂质运动将被追踪成覆盖整个泪液的溶液、去除泪液脂质的泪液和分离的泪液成分的溶液。该研究的成功将导致泪液脂蛋白对异常角膜表面的保护作用的确定，就像干眼症(上皮细胞侵蚀)那样。这些实验是了解干眼症中影响泪膜稳定性和影响眼表的相互作用的关键。特定目的2-研究泪液Lipocalin在溶液中的脂肪酸结合部位。泪液Lipocalin中的配体结合位置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。SUCCESS将提供急需的动态配体结合数据，包括腔内首选的配体位置和三维(3D)运动的阐明。这些数据构成了探索泪液Lipocalin配体相互作用保护泪膜的作用机制的结构基础。具体目标3-进一步阐明调节配体结合的泪液Lipocalin花萼开放端的环中发生的pH诱导的变化。关于台间环AB和GH参与pH驱动机制影响配体结合的假设将被用荧光方法在酸性条件下确定环的位置来检验。脊椎运动、可及性和脂质结合的变化将通过pH滴定进行监测。这些实验将确定调节泪液Lipocalin中配体结合的决定因素，并阐明其中涉及的机制。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，并研究干眼异常表面的这些相互作用，以开发针对这种疾病的有效治疗方法。

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.