BIOGENESIS AND MOLECULAR PATHOGENESIS OF CFTR

囊性纤维化是遗传性蛋白质折叠障碍的原型。它是由囊性纤维化跨膜传导调节因子(CFTR)突变引起的，CFTR是一种在人类上皮细胞顶膜表达的多态蛋白。CFTR的生物发生发生在内质网中，并由一套复杂的细胞机制促进。超过70%的野生型和高达99%的常见突变形式的CFTR不能正确折叠，并被细胞质量控制机制识别，并被泛素-蛋白酶体途径降解。这些观察提出了几个关于CF发病机制和治疗的核心问题。细胞机械如何在不同的细胞隔间协调CFTR折叠？细胞如何识别错误折叠的cftr蛋白？这种认知事件如何与退化联系在一起？如何在患者身上提高CFTR折叠的效率？这项提议的长期目标是表征细胞折叠和质量控制机制的组成、招募和功能，质量控制机制调控新合成的CFTR在内质网中的命运。本研究的具体目的将使用互补的异源表达系统来：L)确定Sec61易位机制在将CFTR组装的早期事件引导到内质网细胞膜中的作用；2)确定决定新合成的CFTR命运的细胞伴侣复合体的动态性质；以及3)研究ER机制在26S蛋白酶体复合体降解CFTR中的作用。拟议的实验将在CFTR内的精确位置加入光活性交联剂探针，以表征定位新生链并将其组装成脂质双层的细胞机械。其他研究将使用体外和体内系统来分析与野生型和突变型CFTR相关的细胞伴侣在成熟和降解的连续阶段中组成的变化。最后，将通过生化互补来鉴定管腔和膜结合的ER质控机。总而言之，这些研究将产生一幅关于细胞机械如何协调和监测多个隔室中的折叠事件，并最终控制生产和非生产途径之间的平衡的全面图景。确定调控这一决策过程的关键成分将是开发药物策略的重要一步，旨在改善患有遗传性疾病(如CF)的患者的突变蛋白的折叠和运输。

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.