CFTR and interacting proteins from shark rectal gland

鲨鱼直肠腺的 CFTR 和相互作用蛋白

• 批准号: 6440235
• 负责人: JOHN R RIORDAN
• 金额: $ 15.7万
• 依托单位: MAYO CLINIC ARIZONA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6440235
• 关键词: chloride channels; crystallization; mass spectrometry; protein isoforms; protein protein interaction; protein purification; protein structure; proteomics; salt gland; sharks; western blottings

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein product of the gene mutated in patients with cystic fibrosis is a member of the largest known class of membrane proteins, the ABC (adenine nucleotide binding cassette) superfamily. However, CFTR is a novel ABC protein as the only one known to be an ion channel. Although there has been considerable debate, it is now generally agreed that the ABC protein structural organization which has been so successful evolutionarily was also adapted in metazoa to form an epithelial chloride channel regulated by phosphorylation and by nucleotide interactions at the characteristic ABC nucleotide binding domains (NBDs). A major impediment to more rapid advances in elucidating the structure‑function relationships of the CFTR protein has been the lack of a rich natural or recombinant source of the protein for purification. The levels of expression achievable in heterolgous mammalian cell expression systems is more than an order of magnitude lower than that of other ABC proteins such as P-glycoprotein for which a low resolution 3-dimensional structure has been obtained by electron diffraction of two dimensional crystals. We have previously purified and reconstituted CFTR expressed from insect cells but a strong tendency to aggregate has hindered attempts to obtain crystalline arrays. We have more recently found that native CFTR can be more readily purified from shark rectal gland, the only accessible solid tissue from which significant amounts of the protein can be obtained. Therefore the first specific aim of the present project is to further explore the feasibility of purifying sufficient quantities of functional CFTR to enable crystallization trials and structure determination. The second related specific aim is to determine the molecular identity of the other larger conductance chloride channel present with CFTR in the apical membranes of the rectal gland tubular cells. This objective will be pursued on the basis of two distinct hypotheses: either that this larger conductance channel may be an isoform of a member of the CIC channel family or that, since it is activated by cyclic AMP, it may interact directly with CFTR. It is possible that both of these hypotheses may hold. However, demonstration of the feasibility of employing this highly-specialized non-mammalian model tissue for either of these purposes should serve to advance understanding of the mechanism whereby CFTR functions and controls chloride secretion.

描述（由申请人提供）： 囊性纤维化跨膜电导调节剂 (CFTR) 糖蛋白 囊性纤维化患者基因突变的产物是 已知最大的一类膜蛋白，ABC（腺嘌呤核苷酸结合蛋白） 盒式磁带）超家族。然而，CFTR 是一种新型 ABC 蛋白，是唯一一种 已知为离子通道。尽管存在相当大的争议，但 现在普遍认为 ABC 蛋白质结构组织具有 在进化上如此成功，也适应了后生动物，形成了 受磷酸化和核苷酸调节的上皮氯离子通道 特征 ABC 核苷酸结合域 (NBD) 处的相互作用。一个 在阐明问题方面取得更快进展的主要障碍 CFTR 蛋白的结构与功能关系一直缺乏 用于纯化的丰富的天然或重组蛋白质来源。这 异源哺乳动物细胞表达中可达到的表达水平 比其他ABC系统低一个数量级以上 蛋白质，例如 P-糖蛋白，其低分辨率 3 维 通过二维电子衍射获得结构 晶体。我们之前已经纯化并重建了从 昆虫细胞，但强烈的聚集倾向阻碍了获得昆虫细胞的尝试 晶体阵列。我们最近发现原生 CFTR 可以更有效 很容易从鲨鱼直肠腺中纯化出来，这是唯一可利用的固体组织 可以获得大量的蛋白质。因此第一个 本项目的具体目的是进一步探讨 纯化足够量的功能性 CFTR 以实现结晶 试验和结构确定。第二个相关的具体目标是 确定其他较大电导氯化物的分子身份 直肠腺管顶膜中存在 CFTR 的通道 细胞。这一目标将基于两个不同的假设来实现： 或者这个较大的电导通道可能是以下成员的同工型 CIC通道家族或者，由于它是由环AMP激活的，所以它可能 直接与 CFTR 互动。这两种假设都有可能成立 抓住。然而，证明使用这种方法的可行性 用于这些目的的高度专业化的非哺乳动物模型组织 应有助于加深对 CFTR 运作机制的理解 并控制氯离子的分泌。