Biogenesis and Molecular Pathogenesis of CFTR

这些研究的长期目标是理解支配正常和病态的一般原则。 CFTR在内质网(ER)膜上折叠。膜蛋白的分子机制 生物发生学是生物学中一个知之甚少的领域，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，遗传突变会导致异常折叠的构象 被细胞质量控制机器迅速识别和降解的物质。现在有证据表明， 在美国30,000名CF患者中，高达90%的原发缺陷是由单一苯丙氨酸缺失引起的 508号位置的残留物。这会导致内质网早期折叠途径的微妙中断，并阻止 膜结合域和胞质结合域的适当结合。理解CF的一个主要限制是 相关的障碍是折叠的许多方面与生物化学复合体中的合成相一致 环境由翻译核糖体和Sec61ER生物合成机器组成。因此， 传统的生化和生物物理工具不太适合研究共翻译折叠事件。 这项提议中的实验将利用最近的发展，现在提供了直接访问 新生多肽在其天然折叠环境中的结构特征。荧光和光活性 探针将被合并到统一的程序性易位中间体队列中，使用合成的 修饰的氨基酰-tRNAs。光交联、荧光猝灭和荧光共振能 然后将使用转移(FRET)来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细胞内的结构特征 多肽控制转位途径，建立跨膜拓扑和膜整合 通过调节新生链暴露于胞液和腔室。第二，我们将确定何时 在合成过程中，以及在易位途径中的哪里，新生的2？结构开始崩溃，以及如何 2)结构形成影响易位浇注动力学。第三，我们将定义共平移折叠 导致3？结构的事件，并决定遗传突变如何破坏CF病的这一过程。 这项工作将有助于我们理解CF的分子发病机制，并为我们提供一个 用于药理学操作生理和病理参数的一般框架 蛋白质折叠障碍。拟议研究与人类健康的相关性： 膜蛋白折叠障碍是一个迅速扩大的医学领域 这影响了数以万计的美国人，付出了巨大的经济和社会代价。 这些疾病的治疗一直受到限制，因为对 生物折叠途径在很大程度上仍然未知。为了克服这个问题，这个 该项目将使用新的生物物理方法来定义何时跨膜片段 在ER生物合成机械的背景下开始折叠，它们是如何插入到 以及遗传性疾病破坏折叠的具体步骤- 相关突变。 。

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 2¿ structure begins to collapse and how 2¿ structure formation influences translocon gating dynamics. Third, we will define cotranaslational folding events that give rise to 3¿ structure 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. Relevance of the proposed research to human health: 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. .