Biomolecular Analysis of Proteins in Visual Disease

描述(申请人提供)：视网膜三磷酸腺苷结合盒(ABC)转运体，ABCR，与几种遗传性视力疾病综合征有关；包括Stargardt病、视锥视杆营养不良、眼底黄斑角膜炎和老年性黄斑变性(ARMD)。遗传分析的概述清楚地表明，错义氨基酸替换在整个ABCR基因的保守区域和非保守区域发生的频率相同。我们的总体目标是研究ABCR蛋白的作用机制，以及Stargardt病和ARMD中观察到的基因突变如何影响视网膜运输中的能量传递，以便未来可能开发出新的更精确的治疗方法。 (1)我们正在用ABCR对核苷酸结合和水解进行详细的结构-功能分析。我们计划测试这一假设，即在Stargardt病和ARMD中观察到的某些突变会导致构象变化，进而影响核苷酸结合和水解。这将有助于我们开发一个预测模型，以预测给定突变对酶活性和相关ABCR结构变化的可能影响。(2)分析底物(视网膜)结合中的胞外区突变。在这个特定的目标中，我们解决了ABCR功能的胞外区突变的生化后果是什么。我们将从几个方向来处理这个重要的问题。我们将研究(A)导致视网膜ATPase刺激丧失的ECD突变的鉴定；(B)重组ECD多肽与视网膜的结合。

DESCRIPTION (provided by applicant): The retinal ATP binding cassette (ABC) transporter, ABCR, is linked to several inherited visual disease syndromes; including Stargardt disease, cone-rod dystrophy, fundus flavimacultitis and age related macular degeneration (ARMD). An overview of genetic analyses clearly demonstrate that missense amino acid substitutions occur with equal frequency in conserved domains, as well as non-conserved domains throughout the whole of the ABCR gene. Our overall aim is to investigate the mechanism of action of ABCR protein and how genetic mutations observed in Stargardt disease and ARMD influence energy transduction in retinal transport so that new and more precise therapies may be developed in the future. (1) We are carrying out a detailed structure-function analysis of nucleotide binding and hydrolysis by ABCR. We plan to test the hypothesis that certain mutations observed in Stargardt disease and ARMD lead to conformational changes which in turn influence nucleotide binding and hydrolysis. This will help us to develop a predictive model for the likely effect of a given mutation on the enzymatic activity and associated structural changes in ABCR. (2) Analyze extracellular domain mutations in substrate (retinal) binding. In this specific aim we address, what are the biochemical consequences of extracellular domain mutations in ABCR function. We will approach this important question from several directions. We will investigate (a) identification of ECD mutations that lead to a loss of retinal stimulation of ATPase; (b) retinal binding by recombinant ECD polypeptides.