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Structural Aspects of Bicarbonate Transport Metabolons

碳酸氢盐转运代谢的结构方面

基本信息

批准号：
6889233
负责人：
Peter M Piermarini
金额：
$ 4.99万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/6889233
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

项目摘要

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运输的机制在肾脏生理学中非常重要。肾小管重新吸收过滤后的HCO3并产生“新的HCO3”，以维持全身酸碱平衡。hco3转运蛋白nbce1和AE1以及碳酸酐酶CA II和CAIV是在肾小管hco3重吸收中起重要作用的蛋白。最近对AE1的研究和NBCel的初步数据表明，这些HCO3转运蛋白可以结合CA II和CA IV并形成HCO3转运代谢产物。该项目的具体目的是利用x射线晶体学来描述1)CA II与hco3转运体之间的细胞内相互作用，以及2)CA IV与hco3转运体之间的细胞外相互作用的结构基础。该方法将生成与AE1和NBCe1的CA- ii和CA- iv结合域对应的重组肽，并将其与各自的CA共结晶。此外，蛋白质-蛋白质相互作用的结合特性将使用固相结合和下拉试验等技术进行表征。了解CAs和hco3转运体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。该研究将提供肾脏HCO3转运机制的关键细节，这将有助于更好地理解所研究蛋白质对肾脏功能的生理学和重要性。