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整合膜蛋白。 UT 在许多哺乳动物组织中高度表达，包括肾脏、肝脏和大脑。 UT 功能在肾脏中最容易理解，UT 对于维持内部髓质区域的高尿素浓度至关重要，以便水分被吸收以产生浓缩尿液。一种 UT 基因被敲除的小鼠表现出吸水能力下降，而人类 UT 的基因变异与血压异常直接相关。这些结果表明 UT 在肾脏生理学和血压调节中发挥着重要作用。然而，目前 UT 结构信息的缺乏阻碍了我们对其架构和功能的理解。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。我们最近从普通脱硫弧菌 (dvUT) 中结晶出一种 UT，它与哺乳动物 UT 具有显着的序列同源性，并且将晶体精炼为衍射间距为 2.3 ¿。我们还启动了 dvUT 的功能研究。我们开发了一种闪烁邻近测定法，首次测量尿素和 UT 之间的平衡结合。我们已成功在非洲爪蟾卵母细胞中表达 dvUT，并发现它介导尿素穿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）也能抑制尿素结合和通过 dvUT 的通量。这些令人兴奋的新结果促使我们提出了一种结构-功能相结合的研究，其目标有以下四个： 目标 1：建立和完善 dvUT 的结构模型。我们将利用与 dvUT 共晶的重原子的反常衍射信号来解决相位问题，并建立和完善 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辨率。目标 2：研究尿素通过 dvUT 转运的机制。尽管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“特征序列”，但dvUT的功能从未被证实。我们将确定 dvUT 是否是功能性尿素转运蛋白，如果是，我们将使用 X 射线晶体学来识别尿素结合位点。尽管许多功能研究表明 UT 通过类似通道的机制进行操作，但并未排除转运机制，因为在某些 UT 中观察到通量率饱和。我们将通过分析结构并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 阻断机制。我们将检查已知的哺乳动物 UT 阻断剂是否影响 dvUT 上的尿素结合和渗透，如果是，我们将通过 X 射线晶体学鉴定阻断剂结合位点。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 dvUT 的结构和功能。目标 4：检查 dvUT 和哺乳动物 UT 之间的尿素渗透和阻断机制是否保守。我们将检查在 dvUT 中观察到的尿素和 UT 阻滞剂的协调是否是通过 UT-A2（一种在肾脏中高表达的哺乳动物 UT）上的同源残基实现的。我们将对 UT-A2 进行突变，并通过卵母细胞通量测定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。总而言之，拟议的研究将大大加深我们对真核和原核 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的三维环境中，我们希望这最终将导致新治疗试剂的开发。 公共健康相关性：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、肝脏、耳朵和睾丸，表明它们在生理学中发挥着重要作用。在人类中，尿素转运蛋白的缺失会导致尿液浓缩能力下降，而尿素转运蛋白的遗传变异与血压变化直接相关。尿素转运蛋白被敲除的小鼠除了尿液浓缩缺陷外，还表现出进行性心脏传导阻滞和青春期提前。因此，尿素转运蛋白是治疗高血压、充血性心力衰竭、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。