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）膜中正常和病理性CFTR折叠的一般原理。膜蛋白生物合成的分子机制代表了一个知之甚少的生物学领域，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，其中遗传突变引起异常折叠的构象异构体，其被细胞质量控制机制快速识别和降解。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位的单个苯丙氨酸残基缺失引起的。这导致ER中早期折叠途径的微妙破坏，并阻止膜结合和胞质结构域的适当结合。理解CF和相关疾病的一个主要限制是，折叠的许多方面与由翻译核糖体和Sec 61 ER生物合成机制组成的生化复杂环境中的合成同时发生。因此，传统的生物化学和生物物理学工具不适合研究共翻译折叠事件。 在这个建议中的实验将利用最近的发展，现在提供直接访问的新生多肽在其天然折叠环境的结构特征。使用合成的修饰的氨酰-tRNA，将荧光和光敏探针掺入到程序化易位中间体的统一队列中。光交联，荧光猝灭和荧光共振能量转移（FRET），然后将用于解决所有天然膜蛋白所面临的三个基本问题。使用野生型和疾病相关的CFTR突变体，我们将首先定义新生多肽内的结构特征如何控制易位途径，并通过调节新生链暴露于胞质和内腔室来建立跨膜拓扑结构和膜整合。第二，我们将确定在合成过程中何时，以及在易位途径内的何处，新生20结构开始崩溃，以及20结构的形成如何影响易位子门控动力学。第三，我们将定义产生30种结构的共翻译折叠事件，并确定遗传突变如何破坏CF疾病的这一过程。这项工作将有助于我们的CF的分子发病机制的理解，并提供了一个通用的框架，其中可重复操作的蛋白质折叠疾病的生理和病理参数。 公共卫生相关性：膜蛋白折叠障碍代表了一个迅速扩大的医学领域，以巨大的经济和社会代价影响成千上万的美国人。对这些疾病的治疗受到限制，因为对生物折叠途径的基本理解仍然很大程度上未知。为了克服这个问题，这个项目将使用新的生物物理方法来定义跨膜片段何时开始在ER生物合成机制的背景下折叠，它们如何插入ER膜，以及折叠被遗传性疾病相关突变破坏的具体步骤。

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.