CFTR--STRUCTURE, FUNCTION, AND DISEASE PATHOGENESIS

CFTR--结构、功能和疾病发病机制

• 批准号: 5213768
• 负责人: BARBARA R GRUBB
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5213768
• 关键词: aldosterone; chloride channels; cystic fibrosis; disease /disorder model; electrophysiology; gene expression; gene mutation; genetically modified animals; human genetic material tag; human tissue; immunocytochemistry; intestines; laboratory mouse; membrane proteins; molecular pathology; monoclonal antibody; mutant; protein sequence; protein structure function; recombinant proteins; sodium channel; tissue /cell culture

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF), the most common genetic disease among Caucasians. The CFTR (1,480 amino acids) appears to be an integral membrane protein, predicted to have 12 transmembrane spanning domains, two cytoplasmically-located nucleotide binding folds and a 240 amino acid "regulatory" (R) domain. Although this protein exhibits homology with several ATP-dependent transporters, recent data suggests that it is intimately involved in chloride channel activity rather than active transport. It is possible that the CFTR possesses an additional, yet undiscovered, activity. The long-term goals of this project are to provide insight into the structure and function of the CFTR and the role of this protein in CF disease pathogenesis. Questions regarding the structure of the CFTR will be addressed by characterizing its membrane topography. These particular experiments will involve the use of specific antibodies to determine the membrane orientation of different regions of the protein, including the putative functional domains (nucleotide binding folds and the R domain). The experiments will be performed on cultured cells expressing recombinant forms of the CFTR. The cellular location of wildtype and mutants of the CFTR will be determined using both the recombinant forms of the protein expressed in culture and the endogenous protein from normal and CF patients. This will ascertain the effect of different CF mutations on CFTR targeting. The function and role of the CFTR in disease pathogenesis will be studied using transgenic mice, made with the CFTR(-/-) mouse. These animals do not express CFTR due to an engineered gene disruption event. Wildtype and mutants of the CFTR will be expressed, as transgenes, in the CFTR(-/- ) mice at different levels by exploring various promoters, as well as by analyzing different founder mice. The transgenic animals will be characterized biochemically, electrophysiologically and clinically. The results of these studies will provide insight into the structure and function of the CFTR and the role that specific mutations play in the course of the disease. Knowledge of the actual physiological abnormality that is responsible for the CF phenotype will aid in the rational design of novel therapeutic strategies.

囊性纤维化跨膜电导调节器中的突变 （CFTR）引起囊性纤维化（CF），这是最常见的遗传疾病 高加索人。 CFTR（1,480个氨基酸）似乎是积分不可分割的 膜蛋白预测具有12个跨膜跨膜结构域， 两个细胞质划分的核苷酸结合褶皱和240个氨基酸 “监管”（R）域。 尽管该蛋白质与 几个依赖ATP的转运蛋白，最近的数据表明它是 密切参与氯化物通道活动，而不是活动 运输。 CFTR可能还具有额外的 未被发现的活动。 该项目的长期目标是 提供有关CFTR和角色的结构和功能的见解 该蛋白在CF疾病发病机理中的蛋白质。 关于 CFTR的结构将通过表征其膜来解决 地形。 这些特殊的实验将涉及 确定不同的膜方向的特定抗体 蛋白质的区域，包括推定的功能域 （核苷酸结合褶皱和R结构域）。 实验将是 在表达CFTR的重组形式的培养细胞上进行。 CFTR的野生型和突变体的细胞位置将是 使用两种表达的蛋白质的重组形式确定 来自正常患者和CF患者的培养和内源性蛋白质。 这 将确定不同CF突变对CFTR靶向的影响。 将研究CFTR在疾病发病机理中的功能和作用 使用CFTR（ - / - ）鼠标制成的转基因小鼠。 这些动物做 由于工程基因中断事件而未表达CFTR。 野生型 CFTR的突变体将作为转基因表示，在CFTR中（ - / - ） ）通过探索各种启动子以及通过 分析不同的创始人小鼠。 转基因动物将是 在生化，电生理和临床上的特征。 这 这些研究的结果将为结构和 CFTR的功能以及特定突变在 疾病的病程。 了解实际生理异常 负责CF表型的原因将有助于理性设计 新型治疗策略。