The (Mis)Folding Pathway of CFTR: Early Interdomain and Protein Interactions

描述（由申请人提供）：囊性纤维化（CF）是一种致命的遗传性疾病，由功能性囊性纤维化跨膜传导调节蛋白（CFTR）缺乏引起。CF无法治愈，该领域的主要目标是开发治疗方法以绕过或纠正CFTR中的基本缺陷。CFTR包含两个跨膜结构域（TMD）、两个核苷酸结合结构域（NBD）和一个调节区（R），其翻译顺序为：TMD 1-NBD 1-R-TMD 2-NBD 2。对于CFTR，结构域各自协同折叠，然后分层缔合以形成功能蛋白。最常见的CF突变是？F508和其他CF突变破坏了该折叠途径中的多个步骤。关于共翻译CFTR（mis）折叠和相互作用的详细信息 细胞蛋白质质量控制机制是有限的。TMD 1和NBD 1中导致CF的突变 在完成CFTR翻译之前使蛋白质不稳定。有人提出，TMD 1和NBD 1在翻译过程中相互作用，这种相互作用被破坏的突变TMD 1或？NBD 1中的F508突变。然而，这些相互作用还没有直接通过实验证明。质量控制蛋白最初何时将突变CFTR识别为错误折叠也是未知的。天然和突变CFTR折叠的翻译和鉴定过程中涉及的蛋白质的机制研究是有针对性的CF治疗的发展所需要的。本提案的目的是：1.测试TMD 1和NBD 1之间的结构域间相互作用在CFTR翻译过程中形成，CF突变破坏这些相互作用的假设。一个强大的技术，旨在网站特异性地将光反应性探针到一个新生的定义长度的链将被用来检查正常和突变CFTR的翻译过程中，这种域间的相互作用。跨膜整合和拓扑结构将与这种相互作用的形成有关。2.鉴定在翻译期间与突变CFTR特异性相互作用的70 kDa蛋白。使用光反应探针方法，已经发现一种未鉴定的70 kDa蛋白，其在翻译早期优先与突变CFTR相互作用。将使用生化分级分离和重构方法鉴定该蛋白质。拟议的研究利用现有的方法在实验室中，以解决CFTR（错误）折叠的基本机制及其与CF的关系，并检查知之甚少的多结构域膜蛋白折叠的过程。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is a lethal genetic disease caused by the lack of functional cystic fibrosis transmembrane conductance regulator protein (CFTR). There is no cure for CF, and the primary goal in the field is to develop therapeutics to bypass or correct the basic defects in CFTR. CFTR contains two transmembrane spanning domains (TMDs), two nucleotide binding domains (NBDs), and one regulatory region (R) that are translated in the order: TMD1-NBD1-R-TMD2-NBD2. For CFTR, domains each fold cotranslationally and then hierarchically associate to form the functional protein. The most common CF mutation, ?F508, and other CF mutations disrupt multiple steps in this folding pathway. Details about cotranslational CFTR (mis)folding and interactions with cellular protein quality control machinery are limited. CF-causing mutations in TMD1 and NBD1 destabilize the protein prior to completion of CFTR translation. It has been proposed that TMD1 and NBD1 interact during translation, and that this interaction is disrupted by either mutations in TMD1 or the ?F508 mutation in NBD1. Yet, these interactions have not been directly demonstrated experimentally. When quality control proteins initially recognize mutant CFTR as misfolded is also not known. Mechanistic studies of native and mutant CFTR folding during translation and identification of the proteins involved are needed for the targeted development of CF therapeutics. The aims of this proposal are to: 1. Test the hypothesis that interdomain interactions between TMD1 and NBD1 are formed during CFTR translation and that CF mutations disrupt these interactions. A powerful technique designed to site-specifically incorporate a photoreactive probe into a nascent chain of defined length will be used to examine this interdomain interaction during translation of normal and mutant CFTR. Transmembrane span integration and topology will be related to formation of this interaction. 2. Identify a 70kDa protein that interacts specifically with the mutant CFTR during translation. Using the photoreactive probe approach, an unidentified 70kDa protein has been found that interacts preferentially with mutant CFTR early during translation. This protein will be identified using a biochemical fractionation and reconstitution approach. The proposed studies utilize extant methodology in the laboratory to address the basic mechanism of CFTR (mis)folding and its relation to CF, and to examine the poorly understood process of multidomain membrane protein folding.