BIOGENESIS AND MOLECULAR PATHOGENESIS OF CFTR

囊性纤维化（CF）是蛋白质折叠遗传性疾病的原型。它是由囊性纤维化跨膜传导调节因子（CFTR）的突变引起的，CFTR是一种在人上皮细胞顶膜中表达的多位蛋白。CFTR的生物发生发生在内质网，并由一套复杂的细胞机制促进。超过70%的野生型和高达99%的常见突变型CFTR不能正常折叠，被细胞质量控制机制识别，并通过泛素-蛋白酶体途径降解。这些观察结果提出了几个关于CF发病机制和治疗的核心问题。细胞机制如何协调不同细胞室中的CFTR折叠？错误折叠的CFTR蛋白如何被细胞识别？这个识别事件是如何与退化耦合的？如何在患者中提高CFTR折叠的效率？本提案的长期目标是表征细胞折叠和质量控制机制的组成、募集和功能，这些机制调节内质网中新合成的CFTR的命运。本研究的具体目的是利用互补的异种表达系统来：1)确定Sec61易位机制在指导CFTR组装进入内质网膜的早期事件中的作用，2)确定控制新合成CFTR命运的细胞伴侣复合物的动态性质，以及3)检查ER机制在26S蛋白酶体复合物降解CFTR中的作用。拟议的实验将在CFTR内的精确位置结合光活性交联探针，以表征细胞机制，定向并将新生链组装成脂质双分子层。进一步的研究将使用体外和体内系统来分析野生型和突变型CFTR在成熟和降解的顺序阶段中与细胞伴侣相关的组成变化。最后，将通过生化互补确定管腔和膜结合的内质网质量控制机制。总之，这些研究将产生一个全面的画面，细胞机制如何协调和监测折叠事件在多个隔室，并最终控制生产和非生产途径之间的平衡。确定调节这一决策过程的关键成分将是开发药理学策略的重要一步，旨在改善CF等遗传性疾病患者突变蛋白的折叠和转运。

Cystic Fibrosis (CF) is a prototype for inherited disorders of protein folding. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a polytopic protein expressed in the apical membrane of human epithelial cells. CFTR biogenesis occurs in the endoplasmic reticulum and is facilitated by a complex set of cellular machinery. Greater than 70% of wild type and up to 99% of common mutant forms of CFTR fail to fold properly and are recognized by cellular quality control machinery and degraded by the ubiquitin- proteasome pathway. These observations raise several questions central to CF pathogenesis and treatment. How does cellular machinery coordinate CFTR folding in different cellular compartments? How is misfolded CFTR protein identified by the cell? How is this recognition event coupled to degradation? And how might the efficiency of CFTR folding be improved in patients? The long term goal of this proposal is to characterize the composition, recruitment and function of cellular folding and quality control machinery that regulates the fate of newly synthesized CFTR in the endoplasmic reticulum. The specific aims of this study will use complimentary heterologous expression systems to: l) define the role of the Sec61 translocation machinery in directing early events of CFTR assembly into the ER membrane, 2) identify the dynamic nature of cellular chaperone complexes that govern the fate of newly synthesized CFTR, and 3) examine the role of ER machinery in CFTR degradation by the 26S proteasome complex. Proposed experiments will incorporate photoactive crosslinking probes at precise locations within CFTR to characterize cellular machinery that orients and assembles the nascent chain into the lipid bilayer. Additional studies will use in vitro and in vivo systems to analyze the changing composition of cellular chaperones associated wild type and mutant CFTR during sequential stages of maturation and degradation. Finally lumenal and membrane-bound ER quality control machinery will be identified by biochemical complementation. Together these studies will generate a comprehensive picture of how cellular machinery coordinates and monitors folding events in multiple compartments and ultimately governs the balance between productive and non-productive pathways. Identification of key components that regulate this decision process will be a major step in the development of pharmacologic strategies aimed at improving folding and trafficking of mutant proteins in patients with inherited disorders such as CF.