MOLECULAR AND CHEMICAL DESCRIPTION OF CFTR FUNCTION

CFTR 功能的分子和化学描述

• 批准号: 2143431
• 负责人: PETER L PEDERSEN
• 金额: $ 16.15万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143431
• 关键词: adenosine triphosphate; antibody; binding proteins; cell membrane; chemical binding; chlorine; circular dichroism; cystic fibrosis; fluorescence spectrometry; gene deletion mutation; genetic manipulation; hydrolysis; molecular cloning; molecular pathology; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; phenylalanine; phosphorylation; protein kinase; protein kinase C; protein purification; protein structure function; proteins

The objectives of this proposal are to complete some very straightforward studies which should greatly facilitate our understanding of both the molecular and chemical basis of cystic fibrosis (CF). The proposed studies will focus on the CFTR protein (cystic fibrosis transmembrane conductance regulator), predicted from cDNA sequence analysis to be 1480 amino acids In length. The predicted protein Includes both a large membrane spanning region and a large cytoplasmic domain, the latter consisting of two putative ATP binding regions (ATP-1, and ATP-11) and a putative regulatory region (R) thought to be a protein kinase target. Significantly, almost 70% of CF patients lack a phenylalanine in the center of the ATP-1 region. Consequently, it has been suggested that the wild type CFTR may be an 'ion motive' ATPase which couples ATP hydrolysis to Cl- transport, and that the phenylalanine deletion mutation may alter the binding or hydrolysis of ATP and, therefore, Cl- transport. With these thoughts in mind, the Specific Aims of this proposal are sixfold: 1.Prepare In large amounts, using both chemical and molecular biological approaches, the two cytoplasmic regions, ATP-1 and ATP-11, of the 'wild type" CFTR protein. 2.Characterize the resultant peptides physically, and assess their capacity to bind and hydrolyze ATP In the presence and absence of Cl-. 3.Prepare as in '1' the ATP-1 region containing the single phenylalanine deletion found in most cystic fibrosis patients. 4.Characterize physically the ATP-1 phenylalanine mutant peptide and assess its capacity to bind and hydrolyze ATP in the presence and absence of Cl-. 5.Prepare, as in "l", a "wild type' and a phenylalanine mutant peptide containing both the regulatory region 'R' and the ATP-1 region, and compare these peptides' capacity to bind and hydrolyze ATP before and after treatment with protein kinases A or C. 6.Assess the effect of antibodies to the ATP-1, ATP-11, and R + ATP-1 regions on the ATPase activity of epithelial cell membranes. The proposed studies are fundamental for understanding the molecular and chemical basis of cystic fibrosis, and may encourage future experiments directed at replacing only a small portion of the "wild type" CFTR gene in CF patients.

这项建议的目的是完成一些非常简单的 这些研究将极大地促进我们对 囊性纤维化（CF）的分子和化学基础。 拟议的研究 将重点关注CFTR蛋白（囊性纤维化跨膜传导 调节子），从cDNA序列分析预测为1480个氨基酸 长的 预测的蛋白质包括一个大的跨膜 区域和一个大的胞质结构域，后者由两个 假定的ATP结合区（ATP-1和ATP-11）和假定的调节区（ATP-11）。 区域（R）被认为是蛋白激酶靶标。 重要的是，几乎 70%的CF患者在ATP-1区域的中心缺乏苯丙氨酸。 因此，已经表明野生型CFTR可能是一种“离子”， 动力性ATP酶，它将ATP水解与Cl-转运偶联， 苯丙氨酸缺失突变可改变ATP的结合或水解 以及氯离子的运输。 带着这些想法，具体 该提案的目标有六个方面： 1.大量制备，使用化学和分子生物学 采用这种方法，“野生型”的两个细胞质区域ATP-1和ATP-11 CFTR蛋白。 2.对所得肽进行物理表征并评估其能力 在Cl-存在和不存在的情况下结合并水解ATP。 3.如“1”中所述制备含有单个苯丙氨酸的ATP-1区域 在大多数囊性纤维化患者中发现缺失。 4.物理表征ATP-1苯丙氨酸突变肽并评估 在Cl-存在和不存在的情况下结合和水解ATP的能力。 5.如“1”中所示，制备“野生型”和苯丙氨酸突变体肽 含有调节区“R”和ATP-1区，并比较 这些肽在前后结合和水解ATP的能力 用蛋白激酶A或C处理。 6.评估抗体对ATP-1、ATP-11和R + ATP-1的影响 上皮细胞膜上ATP酶活性的区域。 所提出的研究是理解分子和 囊性纤维化的化学基础，并可能鼓励未来的实验 目的是仅替换一小部分“野生型”CFTR基因， CF患者