Mechanisms of Polytopic Protein Biogenesis in the ER

内质网多位蛋白生物发生机制

• 批准号: 6331896
• 负责人: WILLIAM R SKACH
• 金额: $ 24.72万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6331896
• 关键词: HeLa cells; Xenopus oocyte; animal tissue; cell free system; chimeric proteins; crosslink; endoplasmic reticulum; laboratory rabbit; membrane biogenesis; membrane proteins; microinjections; molecular assembly /self assembly; molecular chaperones; protein biosynthesis; protein engineering; protein folding; protein isoforms; protein structure function; protein transport; tissue /cell culture; water channel

DESCRIPTION (Adapted from the Applicant's Abstract): The long term goal of this proposal is to establish the molecular basis by which eukaryotic polytopic membrane proteins integrate, fold and assemble in the lipid bilayer of the endoplasmic reticulum (ER). Aquaporins represent a prototype class of polytopic proteins that contain six transmembrane (TM) segments and form selective water-permeable channels in cell membranes. At least six aquaporins are expressed in the mammalian kidney where they play critical roles in fluId and electrolyte homeostasis. While the basic steps of aquaporin assembly in ER have recently been described, very little is known about how cellular machinery mediates specific translocation, membrane integration, and folding events required to establish AQP topology. This is a major limitation in our understanding of normal renal physiology and in particular, pathologic states where aquaporin folding is disrupted, e.g. nephrogenic diabetes insipidus. Recent studies from our laboratory, now provide for the first time, a means to define the molecular interactions responsible for different and novel polytopic protein folding pathways. Because of their significant role in normal physiology, their relatively simple architecture, and their unusual biogenesis mechanisms, aquaporins represent ideal candidates for such a study. The specific aims are: i) to characterize different molecular pathways of aquaporm assembly in the ER membrane, ii) to define how primary structural determinants generate variations in these folding pathways, and iii) to identify novel components within the ER that are required for specialized aspects of aquaporin biogenesis. Proposed experiments will use cell free translation systems to incorporate photoactive crosslinking probes at engineered sites in native, mutant and chimeric aquaporin proteins. These experiments will define molecular interactions between the nascent polypeptide and ER translocation machinery that mediate protein folding and determine how subtle variations in sequence influence normal biogenesis events and topologic outcome. Finally novel factors required for aquaporin maturation will be identified by fractionation and heterologous reconstitution of translocation competent ER membranes that exhibit distinct differences in aquaponn maturation. Together these studies will provide a major advance in our knowledge of the molecular events involved in polytopic protein biogenesis and will establish a foundation for understanding how inherited mutations disrupt biogenesis in human disease.

描述（改编自申请人的摘要）：本项目的长期目标 提议是建立真核多胞体的分子基础 膜蛋白在脂质双层中整合、折叠和组装 内质网（ER）。水通道蛋白代表多胞体的原型类 含有六个跨膜 (TM) 片段并形成选择性的蛋白质 细胞膜上的透水通道。至少有六种水通道蛋白 在哺乳动物肾脏中表达，它们在液体和液体中发挥着关键作用 电解质稳态。虽然 ER 中水通道蛋白组装的基本步骤有 最近被描述，人们对细胞机制如何了解知之甚少 介导特定的易位、膜整合和折叠事件 建立AQP拓扑所需的。这是我们的一个主要限制 了解正常肾脏生理学，特别是病理状态 水通道蛋白折叠被破坏的地方，例如肾性尿崩症。 我们实验室最近的研究首次提供了一种方法 定义导致不同和新颖的多胞体的分子相互作用 蛋白质折叠途径。由于它们在正常情况下发挥着重要作用 生理学、相对简单的结构以及不寻常的生物起源 机制，水通道蛋白是此类研究的理想候选者。 具体目标是： i) 表征不同的分子途径 ER 膜中的 aquaporm 组装，ii) 定义初级结构如何 决定因素在这些折叠途径中产生变化，并且iii) 确定 ER 中专门化所需的新颖组件 水通道蛋白生物发生的各个方面。拟议的实验将使用无细胞 翻译系统结合光敏交联探针 天然、突变和嵌合水通道蛋白中的工程位点。这些 实验将定义新生多肽之间的分子相互作用 和内质网易位机制介导蛋白质折叠并决定如何 序列的细微变化影响正常的生物发生事件和拓扑 结果。最后，水通道蛋白成熟所需的新因素将是 通过易位的分级分离和异源重组来鉴定 在 aquaponn 中表现出明显差异的主管 ER 膜 成熟。这些研究共同将为我们的研究带来重大进展 多位蛋白生物合成中涉及的分子事件的知识和 将为理解遗传突变如何破坏奠定基础 人类疾病的生物发生。