Structure and function of urea transporters

尿素转运蛋白的结构和功能

• 批准号: 8703084
• 负责人: Ming Zhou
• 金额: $ 32.15万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8703084
• 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 Targeting; 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; 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（由申请人提供）：尿素转运蛋白（UT）是促进尿素跨细胞膜转运的膜蛋白。UT在许多哺乳动物组织中高度表达，包括肾、肝和脑。UT功能在肾脏中被最好地理解，其中UT在维持内髓区域中的高尿素浓度中是必不可少的，使得水被吸收以产生浓缩尿。其中一个UT基因被敲除的小鼠表现出吸收水分的能力降低，而人类UT的遗传变异与血压异常直接相关。这些结果表明UT在肾脏生理和调节血压中起重要作用。然而，目前缺乏结构信息的UT阻碍了我们了解他们的架构和功能。因此，我们的长期目标是获得UT促进尿素渗透和UT抑制的原子水平机制。我们最近从与哺乳动物UT具有显著序列同源性的细菌脱硫弧菌（dvUT）中结晶出UT，并将晶体细化到布拉格间距2.3 ½。我们还启动了对dvUT的功能研究。我们已经开发了一种闪烁接近测定法来测量，第一次，尿素和UT之间的平衡结合。我们已经成功地在非洲爪蟾卵母细胞中表达了dvUT，并发现它介导了尿素通过卵母细胞膜的通量。此外，根皮素，一种已知的哺乳动物UT阻断剂，也抑制尿素结合和通过dvUT的通量。这些令人兴奋的新结果使我们提出了一个组合的结构-功能研究，具有以下四个目标：目标1：建立和完善dvUT的结构模型。我们将通过使用来自与dvUT共结晶的重原子的异常衍射信号来解决相位问题，并且我们将建立并改进2.3 <$分辨率的dvUT结构模型。我们将进一步完善结晶条件以提高分离度。目的2：探讨尿素经血管壁转运的机制。虽然dvUT与哺乳动物UT具有高度的序列相似性，并且具有高度保守的UT“签名序列”，但是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射线晶体学来确定尿素结合位点。尽管许多功能研究表明UT通过通道样机制运作，但由于在某些UT中观察到通量率饱和，因此并未排除转运体机制。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3：探讨dvUT阻断的机制。我们将研究已知的哺乳动物UT阻滞剂是否影响尿素结合和渗透dvUT，如果是这样，我们将通过X射线晶体学鉴定阻滞剂结合位点。我们将通过在dvUT上进行点突变来验证结合位点，然后检查突变的dvUT的结构和功能。目的4：研究尿素渗透和阻断机制是否在dvUT和哺乳动物UT之间是保守的。我们将检查在dvUT中观察到的尿素和UT阻断剂的协调是否通过UT-A2上的同源残基实现，UT-A2是一种在肾脏中高度表达的哺乳动物UT。我们将在UT-A2上进行突变，并通过卵母细胞通量测定来检查尿素渗透和阻断。我们还将过表达哺乳动物UT，长期目标是通过X射线晶体学获得高分辨率结构。总之，拟议的研究将大大促进我们对真核和原核UT的理解，并将哺乳动物UT的结构和功能关系置于原子分辨率，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，尿素转运蛋白在多种器官中表达，如肾、脑、心脏、肝、耳和睾丸，表明它们在生理学中起重要作用。在人类中，尿素转运蛋白的丧失会导致浓缩尿液的能力降低，而尿素转运蛋白的遗传变异与血压的变化直接相关。尿素转运蛋白被敲除的小鼠表现出进行性心脏传导阻滞和青春期提前，此外还有浓缩尿液的缺陷。因此，尿素转运蛋白是治疗高血压、充血性心力衰竭和抗利尿激素化合物分泌不当综合征（SIADH）等多种疾病的潜在药物靶点。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。