DEVELOPMENT OF MEMBRANE GLYCOPROTEIN STRUCTURE

本项目的长期目标是了解 膜糖蛋白成熟和折叠的基本机制 变成一个功能性的三维结构。 重要的是，改变 这些过程是几个人类发展的核心。 病理学建议的研究集中在1480个氨基酸的膜上 糖蛋白CFTR。（囊性纤维化跨膜传导调节因子）。 超过90%的囊性纤维化（CF）患者缺乏单一的 CFTR中508位的苯丙氨酸残基。Δ F5 O 8突变 CFTR导致改变的肽片段的热力学稳定性， CFTR在体外和减少量的突变蛋白在顶端 CF上皮细胞膜。因此，CFTR的折叠的描述 它不仅提供了了解这些信息的必要信息， 这种疾病的分子发病机制，但可能提供了一个范例， 了解膜糖蛋白结构的发展。的 该提案的具体目标是： 1.确定野生型的折叠热力学和动力学， CFTR NBD 1的突变形式。光谱方法监测折叠 我们开发的CFTR模型肽将用于测试假设 deltaF 508突变改变了折叠途径，而不是 CFTR的最终天然状态稳定性。 2.阐明伴侣蛋白在CFTR体内折叠中的作用。高 我们实验室的严格免疫沉淀表明，CFTR相互作用 与特定的蛋白质子集，其中一些可能是分子 监护人使用这种方法和酵母双杂交筛选，我们将 鉴定候选分子伴侣并检验差异分子 与分子伴侣的相互作用负责deltaF 508- 内质网中的CFTR。 3.表征这些蛋白质与CFTR相互作用的位点。 本文提供的初步实验表明，CFTR肽 在体外与Hsp 70结合。利用这种生化方法和两种- 混合方法，我们将确定和表征伴侣位点CFTR 并验证囊性纤维化突变和抑制基因 突变可能影响伴侣结合位点。 4.评估改变伴侣和配体水平对CFTR的影响 折页.利用这些系统，我们将测试假设， 分子伴侣和/或配体水平的变化可能允许突变形式的 CFTR以功能性形式进入质膜。 建议的研究对于理解 膜蛋白结构的发展，并可能提供新的 与多种人类疾病有关的信息。

The long range objectives of this project are to understand the fundamental mechanisms by which a membrane glycoprotein matures and folds into a functional three dimensional structure. Significantly, alteration of these processes is central to the development of several human pathologies. The proposed studies focus on the 1480 amino acid membrane glycoprotein CFTR. (cystic fibrosis transmembrane conductance regulator). Greater than 90% of all cystic fibrosis (CF) patients lack a single phenylalanine residue at position 508 in CFTR. The deltaF5O8 mutation of CFTR leads to altered thermodynamic stability of a peptide fragment of CFTR in vitro and decreased amounts of the mutant protein in the apical membrane of CF epithelial cells. Thus, description of the folding of CFTR will not only provide information essential for understanding the molecular pathogenesis of this disease, but may offer a paradigm for understanding the development of membrane glycoprotein structure. The specific aims of the proposal are: 1. DETERMINE THE FOLDING THERMODYNAMICS AND KINETlCS OF WILD TYPE AND MUTANT FORMS OF CFTR NBD1. Spectroscopic methods for monitoring folding of the CFTR model peptides we developed will be used to test the hypothesis that the deltaF508 mutation alters the folding pathway rather than the final native state stability of CFTR. 2. EVALUATE THE ROLE OF CHAPERONE PROTEINS IN CFTR FOLDING IN VIVO. High stringency immunoprecipitations in our laboratory show that CFTR interacts with a specific subset of proteins, some of which may be molecular chaperones. Using this method and a yeast two-hybrid screen we will identify candidate chaperones and test the hypothesis that differential interaction with chaperones is responsible for retention of the deltaF508- CFTR in the endoplasmic reticulum. 3. CHARACTERIZE THE SITES OF INTERACTION OF THESE PROTEINS WITH CFTR. Preliminary experiments presented here demonstrate that a CFTR peptide binds to Hsp70 in vitro. Using this biochemical approach and the two- hybrid method, we will identify and characterize chaperone sites in CFTR and test the hypothesis that cystic fibrosis mutations and suppressor mutations may affect chaperone binding sites. 4. ASSESS THE EFFECT OF ALTERED CHAPERONE AND LIGAND LEVELS ON CFTR FOLDING. Using these systems we will test the hypothesis that alteration of chaperone and/or ligand levels may permit transit of mutant forms of CFTR to the plasma membrane in functional form. The proposed studies are both necessary and fundamental to understanding the development of membrane protein structure, and may provide novel information relevant to several human diseases.