Structure and function of urea transporters

尿素转运蛋白的结构和功能

• 批准号: 7863715
• 负责人: Ming Zhou
• 金额: $ 40.25万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7863715
• 关键词: Address; Affect; Architecture; Bacteria; Binding; Binding Sites; Biological Assay; Blood Pressure; Brain; Cell membrane; Congestive Heart Failure; Crystallization; Defect; Desulfovibrio vulgaris; Development; Diuretics; Drug Delivery Systems; Ear; Equilibrium; Genes; Genetic Variation; Goals; Health; Heart; Heart Block; Human; Hypertension; Inappropriate ADH Syndrome; Integral Membrane Protein; Kidney; Knock-out; Lead; Link; Liver; Mammals; Measures; Mediating; Membrane; Mus; Mutation; Oocytes; Organ; Phase; Phloretin; Physiology; Play; Point Mutation; Principal Investigator; Puberty; Reagent; Research; Resolution; Role; Sequence Homology; Signal Transduction; Structural Models; Structure; Structure-Activity Relationship; Temperature; Testis; Time; Tissues; Urea; Urine; Variant; Vasopressins; Water; X-Ray Crystallography; Xenopus laevis; improved; mutant; novel therapeutics; overexpression; programs; public health relevance; salt balance; urea transporter

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（由适用提供）：尿素转运蛋白（UT）是促进尿素跨细胞膜运输的整体膜蛋白。 UTS在许多哺乳动物组织中高度表达，包括肾脏，肝脏和大脑。在肾脏中最好理解UT功能，在肾脏中，UT对于在髓质区域保持高尿素浓度至关重要，从而吸收水以产生浓度的尿液。敲除ut基因之一的小鼠表现出吸收水的能力降低，并且人UT的遗传变异与血压异常有关。这些结果表明，UT在肾脏生理学和调节血压中起着重要作用。但是，目前缺乏有关UTS的结构信息会阻碍我们对其建筑和功能的理解。因此，我们的长期目标是获得一种原子水平机制，以进行UT准备的尿素渗透和UT抑制作用。最近，我们从desulfovibrio dufgaris（DVUT）的细菌中结晶了一个UT，该UT与哺乳动物UT具有显着的序列同源性，并将晶体改进以衍射为2.3的Bragg间距。我们还开始了关于DVUT的功能研究。我们已经开发了闪烁的接近度评估，以首次测量尿素和UT之间的同等结合。我们已经成功地表达了Laevis卵母细胞中的DVUT，并发现它通过卵母细胞膜介导了尿素通量。此外，菲林蛋白是哺乳动物UTS的已知阻滞剂，还抑制了尿素结合和通过DVUT的通量。这些令人兴奋的新结果使我们提出了一项结合结构功能研究，其目的是：目标1：建立和完善DVUT的结构模型。我们将通过使用与DVUT共结晶的重原子的异常衍射信号来解决相位问题，我们将在2.3»分辨率下构建和完善DVUT的结构模型。我们将进一步完善结晶条件以改善分辨率。目标2：研究通过DVUT运输的机理。尽管DVUT与哺乳动物UT具有很高的序列相似性，并且具有高度构成的UT“签名序列”，但DVUT的功能从未得到证明。我们将确定DVUT是否是功能性尿素转运蛋白，如果是的，我们将使用X射线晶体学识别尿素结合位点。尽管许多功能研究表明，UT通过通道样机制运行，但尚未排除转运蛋白机制，因为在某些UTS对通量速率的满意度中。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。目标3：研究DVUT阻塞的机制。我们将检查是否已知的哺乳动物UT阻滞剂会影响DVUT上的尿素结合和渗透，如果是，我们将通过X射线晶体学识别阻滞剂结合位点。我们将通过在DVUT上进行点突变来验证结合位点，然后检查突变dVuts的结构和功能。目标4：检查尿素渗透和阻滞的机制是否在DVUT和哺乳动物UT之间保守。我们将检查在DVUT中观察到的尿素和UT阻滞剂的协调是否是通过在肾脏中高度表达的哺乳动物UT-A2上的同源残留物来实现的。我们将在UT-A2上进行突变，并通过卵母细胞通量测定法检查尿素渗透和阻断。我们还将过表达哺乳动物UTS，其长期目标是通过X射线晶体学获得高分辨率结构。综上所述，拟议的研究将基本上进一步了解真核和原核UT，并将哺乳动物UT的结构和功能关系在原子分辨率，三维环境中，有时我们希望这会导致新的治疗试剂的发展。 公共卫生相关性：在哺乳动物中，尿素转运蛋白在肾脏，大脑，心脏，肝脏和睾丸等各种器官中表达，这表明它们在生理学中起着重要作用。在人类中，尿素转运蛋白的损失导致浓缩尿液的能力降低，尿素转运蛋白的遗传变异已与血压的变化直接相关。除尿液中的缺陷外，用尿素转运蛋白敲除尿素的小鼠还显示出渐进的心脏块和早期青春期。因此，尿素转运蛋白是治疗各种疾病的潜在药物靶标，从高血压，充血性心力衰竭到抗利尿性马酮化合物（SIADH）不适当分泌的综合征。选择性阻断尿素转运蛋白的化合物将具有利尿作用，但不会干扰盐平衡。