Mechanisms of Polyptopic Protein Biogenesis in the ER

描述(由申请人提供)：该项目的长期目标是确定内质网(ER)膜中水通道蛋白(AQP)整合、折叠和四聚体组装的一般原理和分子机制。水通道蛋白是一个由6个跨度的同源四聚体膜蛋白组成的保守家族，在肾、肺、脑和其他组织的水分动态平衡中起关键作用。水传输的分子基础是通过六个跨膜螺旋和两个半螺旋在单体孔周围以两重倒对称的方式精确排列而实现的。这种结构是由核糖体和Sec61易位机制的协调作用产生的。这一领域的一个主要悬而未决的问题是，新生多肽结构的细微变化如何影响这一机制来指导独特的、通常是病理性的折叠事件。有趣的是，密切相关的水通道蛋白表现出不同的天然折叠途径，这些途径仅由三个变异残基指定。此外，AQP2的遗传点突变破坏了折叠，从而导致肾源性尿崩症(NDI)，这是一种威胁生命的尿液浓度受损的疾病。因此，水通道蛋白是研究膜蛋白生物发生的正常和病理机制的理想模型底物，膜蛋白生物发生与越来越多的人类蛋白质折叠障碍有关。目前的提议将使用修饰的氨基酰tRNA将光交联剂和荧光探针共翻译插入到新生的AQP整合中间体中，这些中间体在合成的特定阶段被动力学捕获。这种方法提供了一种强大的新方法来确定内质网易位机制如何在管腔、胞液和膜环境中协调新生的链折叠，这些环境与细胞内的条件密切相关。通过这些技术，我们将：1)确定不同AQP折叠途径的分子基础，2)准确地定义AQP2折叠在肾源性尿崩症中是如何被破坏的，3)定义AQP四聚化在细胞内转运所需的功能和结构基础。这些研究的结果将极大地促进我们对水通道蛋白生物学的理解，并提高我们理解复杂的整膜蛋白折叠性质的一般能力。他们还将建立一个有用的平台来研究折叠是如何被遗传突变破坏的，从而最终促进治疗各种蛋白质折叠障碍的新策略。 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，因为对于膜蛋白来说，对生物折叠途径的基本了解在很大程度上仍然是未知的。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 this project is to define general principles and molecular mechanisms of Aquaporin (AQP) integration, folding and tetrameric assembly in the endoplasmic reticulum (ER) membrane. Aquaporins comprise a conserved family of 6-spanning, homotetrameric membrane proteins that play critical roles in water homeostasis in the kidney, lung, brain and other tissues. The molecular basis of water transport is achieved by the precise arrangement of six transmembrane helices and two half helices in a two-fold inverted symmetry around a monomeric pore. This structure is generated by the coordinated actions of the ribosome and Sec61 translocation machinery. A major unanswered question in this field is how subtle changes in nascent peptide structure influence this machinery to direct unique, and often pathological folding events. Interestingly, closely related AQPs exhibit different native folding pathways that are specified by just three variant residues. In addition, inherited point mutations in AQP2 disrupt folding and thereby cause nephrogenic diabetes insipidus (NDI), a life threatening disease of impaired urinary concentration. AQPs are therefore ideal model substrates for investigating normal and pathological mechanisms of membrane protein biogenesis that are implicated in a growing number of human protein folding disorders. The current proposal will use modified aminoacyl tRNAs to cotranslationally insert photocrosslinking and fluorescent probes into nascent AQP integration intermediates that are kinetically trapped at defined stages of synthesis. This approach provide a powerful new method to determine how the ER translocation machinery coordinates nascent chain folding in lumenal, cytosolic and membrane environments that closely mimic conditions in the cell. With these techniques we will: 1) define the molecular basis responsible for different AQP folding pathways, 2) define precisely how AQP2 folding is disrupted in nephrogenic diabetes insipidus, 3) define the functional and structural basis of AQP tetramerization required for intracellular trafficking. Results of these studies will significantly advance our understanding of AQP biology and improve our general ability to understand folding properties of complex integral membrane proteins. They will also establish a useful platform to investigate how folding is corrupted by inherited mutations, and thereby ultimately facilitate new strategies to treat diverse 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 remains largely unknown for membrane proteins. 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 specific steps at which folding is disrupted by inherited disease- related mutations.