Biogenesis and Molecular Pathogenesis of CFTR

描述(申请人提供)：这些研究的长期目标是了解支配内质网(ER)膜中正常和病理性CFTR折叠的一般原理。膜蛋白生物发生的分子机制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，这些构象会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，多达90%的原发缺陷是由508位的单个苯丙氨酸残基缺失引起的。这会导致内质网早期折叠途径的微妙中断，并阻止膜结合结构域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，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，折叠的许多方面与合成一致。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，这些发展现在提供了直接进入新生多肽在其天然折叠环境中的结构特征的途径。荧光和光活性探针将被整合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基-tRNA。然后将使用光交联、荧光猝灭和荧光共振能量转移(FRET)来解决所有天然膜蛋白所面临的三个基本问题。利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中的结构特征如何控制转位途径，并通过调节新生细胞链对胞液和管腔的暴露来建立跨膜拓扑和膜整合。其次，我们将确定在合成过程中，新生20结构何时开始崩溃，以及20结构的形成如何影响转位门控动力学。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，并为从药物上操纵蛋白质折叠障碍的生理和病理参数提供一个一般的框架。 与公共卫生相关：膜蛋白折叠障碍是一个迅速扩大的医学领域，影响着数以万计的美国人，造成巨大的经济和社会代价。这些疾病的治疗一直是有限的，因为对生物折叠途径的基本了解在很大程度上仍不清楚。为了解决这一问题，该项目将使用新的生物物理方法来确定跨膜片段在内质网生物合成机制中何时开始折叠，它们如何插入内质网膜，以及折叠被遗传的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.