Structural Aspects of Bicarbonate Transport Metabolons

碳酸氢盐转运代谢的结构方面

• 批准号: 6792429
• 负责人: Peter M Piermarini
• 金额: $ 4.73万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6792429
• 关键词: X ray crystallography; acid base balance; bicarbonates; biological transport; carbonate dehydratase; chemical association; enzyme structure; enzyme substrate; extracellular; intracellular; membrane transport proteins; postdoctoral investigator; protein binding; protein protein interaction; protein purification; protein structure function; recombinant proteins

DESCRIPTION (provided by applicant): Mechanisms responsible for epithelial HCO3-transport are of great importance to renal physiology. The renal tubule reabsorbs filtered HCO3 and generates "new HCO3" in order to maintain systemic acid-base homeostasis. The HCO3-transporters NBCe1& AE1, and carbonic anhydrases CA II & CAIV are proteins that play important roles in HCO3-reabsorption by the renal tubule. Recent studies on AE1, and preliminary data on NBCel, suggest that these HCO3-transporters bind to both CA II and CA IV and form HCO3- transport metabolons. The specific aims of this project are to use X-ray crystallography to describe the structural basis of 1) the intracellular interaction between CA II and the HCO3-transporters, and 2) the extracellular interaction between CA IV and the HCO3-transporters. The approach will be to generate recombinant peptides that correspond to the CA-II and CA-IV binding domains of AE1 and NBCe1 and cocrystallize them with their respective CA. In addition, binding properties of the protein-protein interactions will be characterized using such techniques as solid-phase binding and pull-down assays. Understanding the interactions between CAs and HCO3-transporters will reveal important features of how these proteins interact with one another and may reveal how their interactions influence the function of AE1 and NBCe1.The proposed study will provide key details on the finer mechanisms of renal HCO3 transport, which will provide a better understanding of the physiology and importance of the studied proteins to kidney function.

描述（由申请人提供）： 负责上皮 HCO3 转运的机制对于肾脏生理学非常重要。肾小管重新吸收过滤后的HCO3并产生“新的HCO3”以维持全身酸碱稳态。 HCO3 转运蛋白 NBCe1 和 AE1 以及碳酸酐酶 CA II 和 CAIV 是在肾小管 HCO3 重吸收中发挥重要作用的蛋白质。最近对 AE1 的研究以及 NBCel 的初步数据表明，这些 HCO3 转运蛋白与 CA II 和 CA IV 结合并形成 HCO3 转运代谢物。该项目的具体目标是使用 X 射线晶体学来描述 1) CA II 和 HCO3 转运蛋白之间的细胞内相互作用，以及 2) CA IV 和 HCO3 转运蛋白之间的细胞外相互作用的结构基础。该方法将产生与 AE1 和 NBCe1 的 CA-II 和 CA-IV 结合域相对应的重组肽，并将它们与各自的 CA 共结晶。此外，将使用固相结合和下拉测定等技术来表征蛋白质-蛋白质相互作用的结合特性。了解 CA 和 HCO3 转运蛋白之间的相互作用将揭示这些蛋白质如何相互作用的重要特征，并可能揭示它们的相互作用如何影响 AE1 和 NBCe1 的功能。拟议的研究将提供有关肾脏 HCO3 转运的更精细机制的关键细节，这将有助于更好地了解所研究的蛋白质对肾功能的生理学和重要性。