Mechanisms of Polyptopic Protein Biogenesis in the ER

项目描述（由申请人提供）：本项目的长期目标是明确水通道蛋白（AQP）在内质网（ER）膜中的整合、折叠和四聚体组装的一般原理和分子机制。水通道蛋白包括一个保守的6跨、同四聚体膜蛋白家族，在肾、肺、脑和其他组织的水稳态中起关键作用。水运输的分子基础是通过精确排列六个跨膜螺旋和两个半螺旋在一个单孔周围的双重倒立对称来实现的。这种结构是由核糖体和Sec61易位机制协调作用产生的。这个领域的一个主要未解问题是，新生肽结构的细微变化如何影响这种机制来指导独特的、通常是病理的折叠事件。有趣的是，密切相关的AQPs表现出不同的天然折叠途径，仅由三个变体残基指定。此外，AQP2的遗传点突变破坏折叠，从而导致尿崩症肾病（NDI），这是一种危及生命的尿浓度受损疾病。因此，AQPs是研究与越来越多的人类蛋白质折叠障碍有关的膜蛋白生物发生的正常和病理机制的理想模型底物。目前的提议将使用修饰的氨基酰基trna共翻译插入光交联和荧光探针到新生的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协调腔内、细胞质和膜环境中的新生链折叠，这些环境与细胞中的条件非常相似。通过这些技术，我们将：1)确定负责不同AQP折叠途径的分子基础，2)精确定义肾源性尿崩症中AQP2折叠是如何被破坏的，3)确定细胞内运输所需AQP四聚化的功能和结构基础。这些研究结果将显著促进我们对AQP生物学的理解，并提高我们理解复杂整体膜蛋白折叠特性的一般能力。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疾病的新策略。

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.