Structure and function of urea transporters

尿素转运蛋白的结构和功能

基本信息

  • 批准号:
    8703084
  • 负责人:
  • 金额:
    $ 32.15万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2010
  • 资助国家:
    美国
  • 起止时间:
    2010-06-01 至 2016-05-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Urea transporters (UT) are integral membrane proteins that facilitate transport of urea across cell membrane. UTs are highly expressed in many mammalian tissues, including kidney, liver, and brain. UT function is best understood in the kidney where UT is essential in maintaining a high urea concentration in the inner medullary region so that water is absorbed to produce concentrated urine. Mice with one of the UT genes knocked out show reduced ability to absorb water, and genetic variations of human UT are directly linked to abnormal blood pressure. These results indicate that UT plays an important role in kidney physiology and in regulating blood pressure. However, the current lack of structural information on UTs hampers our understanding of their architecture and function. Our long-term goal is therefore to obtain an atomic level mechanism for UT facilitated urea permeation and UT inhibition. We have recently crystallized a UT from the bacterium Desulfovibrio vulgaris (dvUT) that has significant sequence homology to mammalian UT, and have refined the crystals to diffract to a Bragg spacing of 2.3 ¿. We have also initiated functional studies on dvUT. We have developed a scintillation proximity assay to measure, for the first time, equilibrium binding between urea and UT. We have successfully expressed dvUT in Xenopus laevis oocytes, and found that it mediates urea flux through oocyte membrane. Furthermore, phloretin, a known blocker for mammalian UTs, also inhibits urea binding and flux through dvUT. These exciting new results have led us to propose a combined structure- function study with the following four aims: Aim 1: To build and refine a structural model of dvUT. We will solve the phase problem by using anomalous diffraction signals from heavy atoms co-crystallized with dvUT, and we will build and refine a structural model of dvUT at 2.3 ¿ resolution. We will further refine crystallization conditions to improve the resolution. Aim 2: To investigate mechanism of urea transport through dvUT. Although dvUT has high sequence similarity to mammalian UTs, and has the highly conserved UT "signature sequence", the function of dvUT has never been demonstrated. We will determine if dvUT is a functional urea transporter, and if so, we will then use X-ray crystallography to identify urea binding sites. Although many functional studies suggest that UT operates by a channel-like mechanism, the transporter mechanism has not been ruled out because in certain UTs saturation of flux rate is observed. We will address this question by analyzing the structure and by measuring urea flux at different temperatures. Aim 3: To investigate mechanism of dvUT blockade. We will examine if known mammalian UT blockers affect urea binding and permeation on dvUT, and if so, we will identify blocker binding sites by X-ray crystallography. We will verify the binding sites by making point mutations on dvUT, and then examine both the structure and function of mutant dvUTs. Aim 4: To examine if the mechanisms of urea permeation and blockade are conserved between dvUT and mammalian UT. We will examine whether coordination of urea and UT blockers observed in dvUT is achieved by homologous residues on UT-A2, a mammalian UT that is highly expressed in kidney. We will make mutations on UT-A2, and examine urea permeation and blockade by an oocyte flux assay. We will also overexpress mammalian UTs with the long term goal of obtaining a high resolution structure by X-ray crystallography. Taken together, the proposed research will substantially further our understanding of both eukaryotic and prokaryotic UT, and will place structure and function relationships of mammalian UT in an atomic-resolution, three-dimensional context which eventually we hope will lead to the development of new therapeutic reagents. PUBLIC HEALTH RELEVANCE: In mammals, urea transporters are expressed in a wide array of organs, such as kidney, brain, heart, liver, ear, and testis, suggesting that they play an important role in physiology. In humans, loss of urea transporter causes reduced capability to concentrate urine, and genetic variations of urea transporters have been directly linked to variations in blood pressures. Mice with urea transporters knocked out showed progressive heart block and early puberty, in addition to defects in concentrating urine. Therefore, urea transporter is a potential drug target for treating a variety of conditions ranging from hypertension, congestive heart failure, to syndrome of inappropriate secretion of antidiuretic hormone compounds (SIADH). Compounds that selectively block urea transporter will have diuretic effect but will not interfere with the salt balance.
描述(由申请人提供):尿素转运蛋白(UT)是一种完整的膜蛋白,可促进尿素跨细胞膜的转运。UT在许多哺乳动物组织中高度表达,包括肾脏、肝脏和脑。UT的功能在肾脏得到最好的理解,在肾脏中,UT是维持内髓区域高尿素浓度的关键,从而使水被吸收以产生浓缩的尿液。其中一个UT基因被敲除的老鼠表现出吸收水分的能力降低,而人类UT的基因变异与异常血压直接相关。这些结果表明,UT在肾脏生理和调节血压方面起着重要作用。然而,目前关于UTS的结构信息的缺乏阻碍了我们对其体系结构和功能的理解。因此,我们的长期目标是获得一种原子水平的UT促进尿素渗透和UT抑制的机制。我们最近从普通脱硫弧菌(DvUT)中分离出一种与哺乳动物UT有显著序列同源性的UT,并对晶体进行了提纯,使其衍射峰的布拉格间距为2.3?我们还启动了dvUT的功能研究。我们首次开发了一种闪烁邻近分析法来测量尿素和UT之间的平衡结合。我们已经成功地在非洲爪哇卵母细胞中表达了dvUT,并发现它通过卵母细胞膜调节尿素的转运。此外,已知的哺乳动物尿路连接素阻滞剂,也抑制尿素结合和通过dvUT的通量。这些令人振奋的新结果促使我们提出了一项结构-功能相结合的研究,主要有以下四个目标:目标1:建立和完善dvUT的结构模型。我们将利用与dvUT共结晶的重原子的反常衍射信号来解决位相问题,并将建立和完善2.3?分辨率的dvUT结构模型。我们将进一步细化结晶条件,以提高分辨率。目的2:探讨尿素通过dvUT的转运机制。虽然dvUT与哺乳动物的UT有很高的序列相似性,并且具有高度保守的UT“签名序列”,但dvUT的功能从未被证实。我们将确定dvUT是否是一种功能性尿素转运蛋白,如果是,我们将使用X射线结晶学来确定尿素结合部位。虽然许多功能研究表明,UT是通过一种类似通道的机制工作的,但仍未排除转运机制,因为在某些UTS中观察到了通量速率的饱和。我们将通过分析结构和测量不同温度下的尿素通量来解决这个问题。目的:探讨dvUT的阻断机制。我们将检查已知的哺乳动物UT阻滞剂是否影响dvUT上的尿素结合和渗透,如果是,我们将通过X射线结晶学确定阻滞剂结合部位。我们将通过在dvUT上进行点突变来验证结合位点,然后检测突变的dvUT的结构和功能。目的:探讨DVUT和哺乳动物UT之间尿素渗透和阻断的机制是否保守。我们将检查在dvUT中观察到的尿素和UT阻滞剂的协调是否通过UT-A2上的同源残基实现,UT-A2是一种在肾脏高表达的哺乳动物UT。我们将对UT-A2进行突变,并通过卵母细胞通量试验检测尿素的渗透和阻断。我们还将过度表达哺乳动物的UTS,长期目标是通过X射线结晶学获得高分辨率的结构。综上所述,拟议的研究将大大加深我们对真核和原核生物UT的理解,并将把哺乳动物UT的结构和功能关系置于原子分辨的三维背景下,最终我们希望这将导致新的治疗试剂的开发。 公共卫生相关性:在哺乳动物中,尿素转运蛋白在广泛的器官中表达,如肾脏、脑、心脏、肝脏、耳朵和睾丸,这表明它们在生理上发挥着重要作用。在人类中,尿素转运蛋白的缺失会导致浓缩尿液的能力降低,而尿素转运蛋白的基因变异与血压的变化直接相关。尿素转运蛋白被敲除的小鼠出现进行性心脏传导阻滞和青春期提前,此外还出现了尿液浓缩缺陷。因此,尿素转运体是一种潜在的药物靶点,用于治疗从高血压、充血性心力衰竭到抗利尿激素化合物分泌不当综合征(SIADH)等各种疾病。选择性阻断尿素转运蛋白的化合物将具有利尿作用,但不会干扰盐分平衡。

项目成果

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Ming Zhou其他文献

Analysis of Performance of Suspended Pre-stressed Steel Shells with Large Span in Fire
大跨度悬吊预应力钢壳火灾性能分析
  • DOI:
  • 发表时间:
    2011
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Xintang Wang;Jie Yin;Ming Zhou;Zhiguo Xie
  • 通讯作者:
    Zhiguo Xie

Ming Zhou的其他文献

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{{ truncateString('Ming Zhou', 18)}}的其他基金

Structure and Function of a Phosphorylation Coupled Saccharide Transporter
磷酸化偶联糖转运蛋白的结构和功能
  • 批准号:
    8317627
  • 财政年份:
    2011
  • 资助金额:
    $ 32.15万
  • 项目类别:
Structure and Function of a Phosphorylation Coupled Saccharide Transporter
磷酸化偶联糖转运蛋白的结构和功能
  • 批准号:
    8669013
  • 财政年份:
    2011
  • 资助金额:
    $ 32.15万
  • 项目类别:
Structure and Function of a Phosphorylation Coupled Saccharide Transporter
磷酸化偶联糖转运蛋白的结构和功能
  • 批准号:
    8160526
  • 财政年份:
    2011
  • 资助金额:
    $ 32.15万
  • 项目类别:
Structure and Function of a Phosphorylation Coupled Saccharide Transporter
磷酸化偶联糖转运蛋白的结构和功能
  • 批准号:
    8637312
  • 财政年份:
    2011
  • 资助金额:
    $ 32.15万
  • 项目类别:
Structure and Function of a Phosphorylation Coupled Saccharide Transporter
磷酸化偶联糖转运蛋白的结构和功能
  • 批准号:
    8475630
  • 财政年份:
    2011
  • 资助金额:
    $ 32.15万
  • 项目类别:
Structure and function of urea transporters
尿素转运蛋白的结构和功能
  • 批准号:
    7863715
  • 财政年份:
    2010
  • 资助金额:
    $ 32.15万
  • 项目类别:
Structural Genomics and Membrane Proteins
结构基因组学和膜蛋白
  • 批准号:
    8151974
  • 财政年份:
    2010
  • 资助金额:
    $ 32.15万
  • 项目类别:
Structure and function of urea transporters
尿素转运蛋白的结构和功能
  • 批准号:
    8473209
  • 财政年份:
    2010
  • 资助金额:
    $ 32.15万
  • 项目类别:
Pilot 1: Investigating Conformational Changes in Saccharide Transporter
试点 1:研究糖转运蛋白的构象变化
  • 批准号:
    8933663
  • 财政年份:
    2010
  • 资助金额:
    $ 32.15万
  • 项目类别:
Structure and function of urea transporters
尿素转运蛋白的结构和功能
  • 批准号:
    8277405
  • 财政年份:
    2010
  • 资助金额:
    $ 32.15万
  • 项目类别:

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