Biogenesis and Molecular Pathogenesis of CFTR

描述（由申请人提供）：这些研究的长期目标是了解内质网（ER）膜中正常和病理CFTR折叠的一般原理。膜蛋白生物发生的分子机制是一个鲜为人知的生物学领域，对人类健康和疾病有重大影响。囊性纤维化（CF）就是这样一个例子，遗传突变引起异常折叠的构象，这些构象被细胞质量控制机制迅速识别和降解。现在有证据表明，在美国30,000名CF患者中，高达90%的原发性缺陷是由508位单个苯丙氨酸残基的缺失引起的。这导致内质网早期折叠途径的微妙破坏，并阻止膜结合和细胞质结构域的适当结合。了解CF和相关疾病的一个主要限制是，折叠的许多方面是在由翻译核糖体和Sec61 ER生物合成机制组成的生化复杂环境中与合成同时发生的。因此，传统的生物化学和生物物理工具不适合研究共翻译折叠事件。

DESCRIPTION (provided by applicant): The long term goal of these studies is to understand general principles that govern normal and pathological CFTR folding in the endoplasmic reticulum (ER) membrane. Molecular mechanisms of membrane protein biogenesis represent a poorly understood area of biology with major implications for human health and disease. Cystic fibrosis (CF) is one such example where inherited mutations give rise to abnormally folded conformers that are rapidly recognized and degraded by cellular quality control machinery. Evidence now indicates that the primary defect in up to 90% of the 30,000 CF patients in the US is caused by deletion of a single phenylalanine residue at position 508. This causes a subtle disruption of the early folding pathway in the ER and prevents proper association of membrane-bound and cytosolic domains. A major limitation in understanding CF and related disorders is that many aspects of folding occur coincident with synthesis in a biochemically complex environment comprised of the translating ribosome and the Sec61 ER biosynthetic machinery. Therefore, traditional biochemical and biophysical tools are poorly suited to study cotranslational folding events. Experiments in this proposal will take advantage of recent developments that now provide direct access to structural features of the nascent polypeptide in its native folding environment. Fluorescent and photoactive probes will be incorporated into uniform cohorts of programmed translocation intermediates using synthetic modified aminoacyl-tRNAs. Photocrosslinking, fluorescence quenching and fluorescence resonance energy transfer (FRET) will then be used to address three fundamental problems faced by all native membrane proteins. Using wild type and disease related CFTR mutants, we will first define how structural features within the nascent polypeptide control the translocation pathway and establish transmembrane topology and membrane integration by regulating nascent chain exposure to cytosolic and lumenal compartments. Second, we will determine when during synthesis, and where within the translocation pathway, nascent 20 structure begins to collapse and how 20 structure formation influences translocon gating dynamics. Third, we will define cotranslational folding events that give rise to 30 structures and determine how inherited mutations disrupt this process in CF disease. This work will contribute significantly to our understanding of the molecular pathogenesis of CF and provide a general framework with which to pharmacologically manipulate physiological and pathological parameters of protein folding disorders. PUBLIC HEALTH RELEVANCE: Disorders of membrane protein folding represent a rapidly expanding area of medicine that affects tens of thousands of Americans at enormous economic and social cost. Treatments for these disorders have been limited because basic understanding of biological folding pathways remain largely unknown. To overcome this problem, this project will use novel biophysical approaches to define when transmembrane segments begin to fold in the context of ER biosynthetic machinery, how they are inserted into the ER membrane, and the specific steps at which folding is disrupted by inherited disease- related mutations.