Mechanisms of Polyptopic Protein Biogenesis in the ER

描述（由申请人提供）：本项目的长期目标是确定水通道蛋白（AQP）在内质网（ER）膜中整合、折叠和四聚体组装的一般原理和分子机制。水通道蛋白是一个保守的6跨同源四聚体膜蛋白家族，在肾、肺、脑和其他组织中的水稳态中起关键作用。水运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双重反向对称的精确排列来实现的。这种结构是由核糖体和Sec61易位机制的协调作用产生的。在这个领域中一个主要的未回答的问题是，新生肽结构的微妙变化如何影响这种机制，以指导独特的，往往是病理性的折叠事件。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中的遗传性点突变破坏折叠，从而引起肾源性尿崩症（NDI），这是一种危及生命的尿浓度受损的疾病。因此，水通道蛋白是理想的模型基板，用于研究膜蛋白生物合成的正常和病理机制，涉及越来越多的人类蛋白质折叠疾病。目前的建议将使用修饰的氨酰tRNA协同插入光交联和荧光探针到新生的AQP整合中间体，在合成的定义阶段动力学捕获。这种方法提供了一种强大的新方法来确定ER易位机制如何在紧密模拟细胞条件的内腔、胞质和膜环境中协调新生链折叠。通过这些技术，我们将：1）定义负责不同AQP折叠途径的分子基础，2）精确定义在肾性尿崩症中AQP 2折叠如何被破坏，3）定义细胞内运输所需的AQP四聚体的功能和结构基础。这些研究的结果将显着推进我们的理解AQP生物学和提高我们的一般能力，了解复杂的完整膜蛋白的折叠特性。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，从而最终促进治疗不同蛋白质折叠疾病的新策略。 公共卫生相关性：膜蛋白折叠障碍代表了一个迅速扩大的医学领域，以巨大的经济和社会代价影响成千上万的美国人。这些疾病的治疗受到限制，因为对膜蛋白的生物折叠途径的基本理解仍然是未知的。该项目将使用新的生物物理方法来定义跨膜片段何时开始在ER生物合成机制的背景下折叠，它们如何插入ER膜，以及折叠被遗传性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.