Biogenesis and Molecular Pathogenesis of CFTR

这些研究的长期目标是了解正常和病理的一般原则 CFTR在内质网（ER）膜中折叠。膜蛋白的分子机制 生物发生是对人类健康和疾病有重大影响的生物学领域知之甚少。 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 它们被细胞质量控制机制迅速识别和降解。现在的证据表明， 在美国30，000名CF患者中，高达90%的原发性缺陷是由单个苯丙氨酸缺失引起的 在位置508处的残基。这会导致ER中早期折叠途径的微妙中断， 膜结合域和胞质域的适当结合。理解CF的一个主要限制， 与此相关的疾病是，折叠的许多方面与生物化学复合物中的合成同时发生。 翻译环境由翻译核糖体和Sec 61 ER生物合成机制组成。因此，我们认为， 传统的生物化学和生物物理学工具不太适合研究共翻译折叠事件。 该提案中的实验将利用最近的发展，现在可以直接访问 新生多肽在其天然折叠环境中的结构特征。荧光和光敏 探针将被掺入到程序化易位中间体的统一队列中， 修饰的氨酰-tRNA。光交联、荧光猝灭和荧光共振能 然后将使用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CFTR中的结构特征如何影响CFTR的表达。 多肽控制转运途径并建立跨膜拓扑结构和膜整合 通过调节新生链暴露于胞质和内腔室。第二，我们将确定何时 在合成过程中，以及在易位途径中，新生的2？结构开始崩溃，以及如何 2结构形成影响易位门控动力学。第三，我们将定义共转换折叠 这些事件引起3 <$结构，并决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对CF的分子发病机制的理解，并提供一个新的研究方向。 一个通用的框架，利用该框架可以对患者的生理和病理参数进行手动操作， 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展的医学领域 这影响了成千上万的美国人，付出了巨大的经济和社会代价。 对这些疾病的治疗一直是有限的，因为对这些疾病的基本理解是， 生物学折叠途径仍然是未知的。为了解决这个问题，这 该项目将使用新的生物物理方法来定义何时跨膜片段 开始折叠的背景下，ER生物合成机制，他们是如何插入到 ER膜，以及折叠被遗传性疾病破坏的具体步骤- 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. .