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

CFTR 的（错误）折叠途径：早期域间和蛋白质相互作用

• 批准号: 8042527
• 负责人: Anna Elizabeth Patrick
• 金额: $ 3.23万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8042527
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Address; Age; Alleles; American; Amino Acids; Binding; Biochemical; Bypass; Cell Membrane Proteins; Cell membrane; Cellular Membrane; Cessation of life; Chloride Ion; Chlorides; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Development; Disease; Endoplasmic Reticulum; Epithelium; Foundations; Fractionation; Gene Proteins; Goals; Health; Hereditary Disease; Induced Mutation; Integral Membrane Protein; Knowledge; Laboratories; Length; Longevity; Lung diseases; Membrane; Membrane Proteins; Methodology; Mucous body substance; Mutate; Mutation; Nucleic Acid Regulatory Sequences; Nucleotides; Pathology; Pathway interactions; Patients; Pharmacologic Substance; Phenylalanine; Physiological; Positioning Attribute; Process; Proteins; Quality Control; Quality of life; Recurrence; Reproductive system; Respiratory System; Site; System; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Thick; Translating; Translations; United States; Work; design; experience; gastrointestinal system; member; mutant; palliative; prevent; protein folding; protein function; protein misfolding; protein structure; reconstitution; repaired; therapeutic development; therapeutic target

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.

描述(申请人提供)：囊性纤维化(CF)是一种由功能性囊性纤维化跨膜传导调节蛋白(CFTR)缺乏引起的致死性遗传性疾病。目前还没有治愈CF的方法，该领域的主要目标是开发治疗方法，绕过或纠正CFTR的基本缺陷。CFTR包含两个跨膜跨膜结构域(TMDs)、两个核苷酸结合结构域(NBD)和一个调节区(R)，其翻译顺序为：TMD1-NBd1-R-TMD2-Nbd2。对于CFTR域，每个结构域都以共翻译的方式折叠，然后分级结合形成功能蛋白。最常见的Cf突变？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(错位)折叠和相互作用的详细信息 与细胞蛋白质质量控制机制是有限的。Cf导致TMD1和NBD1的突变 在cftr翻译完成之前破坏蛋白质的稳定性。有人认为TMD1和NBD1在翻译过程中相互作用，这种相互作用被TMD1的突变或NBD1的F508突变所破坏。然而，这些相互作用还没有得到直接的实验证明。质量控制蛋白最初何时识别突变的cftr为错误折叠也是未知的。对天然和突变的CFTR在翻译过程中折叠的机制进行研究，并鉴定所涉及的蛋白质，对于CF治疗的靶向开发是必要的。本建议的目的是：1.验证TMD1和NBD1之间的结构域间相互作用是在CFTR翻译过程中形成的假设，并且CF突变破坏了这些相互作用。一种强大的技术旨在将光反应探针定点结合到定义长度的新生链中，将用于检测正常和突变CFTR翻译过程中的这种结构域间相互作用。跨膜跨度整合和拓扑结构将与这种相互作用的形成有关。2.鉴定一个70 kDa的蛋白，该蛋白在翻译过程中与突变的cftr发生特异性的相互作用。利用光反应探针方法，发现了一个未知的70 kDa蛋白质，它在翻译早期优先与突变的CFTR相互作用。这种蛋白质将使用生化分离和重组的方法进行鉴定。这些研究利用实验室现有的方法学来研究CFTR(Mis)折叠的基本机制及其与CF的关系，并研究了人们对多结构域膜蛋白折叠过程知之甚少的过程。