DEVELOPMENT OF MEMBRANE GLYCOPROTEIN STRUCTURE

该项目的长期目标是了解 膜糖蛋白成熟和折叠的基本机制 变成了一个功能性的三维结构。值得注意的是，改变 这些过程中的核心是几个人类的发展 病理学。建议的研究重点是1480氨基酸膜。 糖蛋白cftr。(囊性纤维化跨膜电导调节剂)。 超过90%的囊性纤维化(CF)患者缺乏单一 CFTR508位的苯丙氨酸残基。的DeltaF5O8突变 Cftr导致多肽片段热力学稳定性改变 CFTR在体外和根尖突变型蛋白的减少 CF上皮细胞的细胞膜。因此，对CFTR折叠的描述 不仅提供了理解 这种疾病的分子发病机制，但可能提供一种范例 了解膜糖蛋白结构的发育。这个 该提案的具体目标是： 1.测定野生型和野生型的折叠热力学和KINETLCS Cftr nbd1的突变形式。光谱学方法监测折叠的研究 我们开发的CFTR模型多肽将被用来检验这一假设 DeltaF508突变改变了折叠途径，而不是 CFTR的最终本征态稳定性。 2.研究伴侣蛋白在体内CFTR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 具有特定的蛋白质子集，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 确定候选伴侣并检验区分的假设 与伴侣的相互作用是DeltaF508保留的原因- 内质网中的CFTR。 3.鉴定这些蛋白质与CFTR的相互作用部位。 这里提供的初步实验表明，CFTR多肽 在体外与Hsp70结合。使用这种生化方法和两种- 混合方法，我们将识别和表征CFTR中的伴侣位点 并测试囊性纤维化突变和抑制子的假设 突变可能会影响伴侣结合位点。 4.评估伴侣和配体水平改变对CFTR的影响 折叠。使用这些系统，我们将检验这样一种假设 伴侣和/或配基水平的变化可能允许突变形式的 Cftr以功能形式传递到质膜。 拟议的研究对于理解这些问题既是必要的，也是基本的。 膜蛋白结构的发展，并可能提供新的 与几种人类疾病相关的信息。

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.