Biomolecular Analysis of Proteins in Visual Disease

描述（由申请人提供）：ABCA4基因编码视网膜特异性ATP结合盒（ABC）转运蛋白，ABCR，定位于视杆和视锥细胞感光器外段。ABCR被认为在视网膜衍生物通过光感受器膜的运输中起作用。ABCA4基因突变可导致几种遗传性视网膜变性（RD）；包括Stargardt病、锥杆营养不良（arCRD）、黄斑眼底（FFM）和年龄相关性黄斑变性（ARMD）。这些变性代表了广泛的临床表型，所有这些都与脂褐素的积累有关，这被认为部分是由于ABCR运输缺陷。我们的总体目标是研究ABCR蛋白的作用机制，以及在Stargardt病和其他rd中观察到的基因突变如何影响ABCR的结构和功能，以便在不久的将来开发出新的更精确的治疗方法。我们提出的研究将集中在两个方面，首先是ABCR特定结构域的功能，其次是整个蛋白质的功能。我们计划验证细胞外（ECD）或核苷酸结合（NBD）结构域RD相关突变导致构象变化的假设，构象变化反过来影响蛋白质-蛋白质或蛋白质-配体相互作用。这将有助于我们建立一个预测模型，预测给定突变对这些结构域的结构和功能变化的可能影响。其次，我们将检验视网膜或视网膜衍生物在膜结合环境中直接与ABCR结构域相互作用的假设。为了验证这一假设，我们将直接在膜囊中表达全长ABCR。这些研究将为我们研究RD相关突变对整个ABCR分子的影响提供一条途径。在美国，黄斑变性是导致失明的主要原因，视力障碍是一个日益严重的公共卫生问题，特别是随着人口老龄化。几种影响年轻人和老年人的变性都与ABCR基因的突变有关。这些研究将有助于更准确地了解ABCR在黄斑变性（如锥杆营养不良、Stargardt病和年龄相关性黄斑变性）发展中的作用。这将使我们能够对那些ABCR基因突变的个体进行更准确的预后，并最终开发出更好的治疗方法。总的来说，我们的研究试图将通过临床遗传学获得的知识和这些突变在分子水平上的后果联系起来，从而使我们能够建立临床基因型-生化表型相关性。

DESCRIPTION (provided by applicant): The ABCA4 gene encodes a retina specific ATP binding cassette (ABC) transporter, ABCR, which is localized to the rod and cone photoreceptor outer segments. ABCR is thought to function in the transport of retinal derivatives across photoreceptor disk membranes. Mutations in the ABCA4 gene lead to several inherited forms of retinal degeneration (RD); including Stargardt disease, cone-rod dystrophy (arCRD), fundus flavimaculatus (FFM) and age related macular degeneration (ARMD). These degenerations represent a broad spectrum of clinical phenotypes, all associated with accumulations of lipofuscin, which is thought to be in part due to defects in transport by ABCR. Our overall aim is to investigate the mechanism of action of ABCR protein and how genetic mutations observed in Stargardt disease and other RDs influence ABCR structure and function so that new and more precise therapies could be developed in the near future. Our proposed studies will focus on two areas, first the function of specific domains of ABCR and second the function of the protein as a whole. We plan to test the hypothesis that RD associated mutations, in the extracellular (ECD) or nucleotide binding (NBD) domains lead to conformational changes, which in turn influence protein-protein or protein-ligand interactions. This will help us to develop a predictive model for the likely effect of a given mutation on structural and functional changes in these domains. Second, we shall test the hypothesis that retinal or retinal derivatives interact directly with the domains of ABCR in a membrane bound environment. We shall express full-length ABCR directly in membrane vesicles in order to test this hypothesis. These studies will provide us an avenue to investigate the effects of RD associated mutations on the ABCR molecule as a whole. Macular degenerations are the leading cause of blindness in the United States, and visual impairment is an increasing public health issue, particularly with our aging population. Several types of degeneration, affecting both the young and the old, have been associated with mutations in the ABCR gene. These studies will lead to a more accurate understanding of the involvement ABCR in the development of macular degenerations, such as Cone Rod Dystrophy, Stargardt disease, and Age Related Macular Degeneration. This will enable us to develop a more accurate prognosis of those individuals identified with mutations in the ABCR gene, and ultimately, the development of better therapies. Overall, our studies seek to bridge the knowledge obtained through clinical genetics, and the consequences of these mutations at the molecular level -- thereby enabling us to develop a clinical genotype-biochemical phenotype correlation.