Structural and functional studies of urea channels

尿素通道的结构和功能研究

• 批准号: 8019537
• 负责人: THOMAS WALZ
• 金额: $ 21.55万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8019537
• 关键词: AQP9 gene; Actinobacillus pleuropneumoniae; Address; Adipocytes; Amides; Antibiotics; Arsenic; Arsenic Poisoning; Arsenites; Biochemical; Biological; Boston; Cells; Characteristics; Collaborations; Crystallization; Crystallography; Data; Data Set; Development; Diuretics; E coli GlpF protein; Eating Disorders; Electron Microscopy; Electrons; Eukaryota; Family; Fasting; Gluconeogenesis; Glycerol; Goals; Helicobacter pylori; Homologous Gene; Homology Modeling; Human; Image; Individual; Ingestion; Insecta; Israel; Kinetics; Lead; Liposomes; Liver; Mammals; Measurement; Mediating; Medical center; Membrane; Membrane Proteins; Microbe; Microscopic; Modeling; Molecular; Names; Nitrogen; Pattern; Phase; Physiological; Play; Principal Investigator; Proteins; Rattus; Recombinant Proteins; Recombinants; Research; Resolution; Role; Sequence Homology; Source; Specificity; Specimen; Structure; Structure-Activity Relationship; Substrate Specificity; Techniques; Testing; Toxic effect; Transport Process; Urea; Uropathogenic E. coli; Water; Work; X-Ray Crystallography; abstracting; base; electron crystallography; improved; inhibitor/antagonist; interest; member; novel; pathogen; programs; protein function; proteoliposomes; reconstitution; research study; salt balance; solute; tool; two-dimensional; urea transporter; water channel

Abstract Urea is the main catabolite in mammals and an important nitrogen source for many microbes. This proposal focuses on structural and functional studies of membrane proteins that facilitate transmembrane urea transport, specifically members of the aquaporin (AQP), urea transporter (UT), and urea/amide channel (UAC) families. We are studying AQP9, which has the broadest substrate specificity among all known AQPs, UreI from Helicobacter pylori, a member of the UAC family, and the urea transporters UT-Apl from Actinobacillus pleuropneumoniae and UT-Ec from the uropathogenic E. coli strain 536. The Specific Aims of this proposal are: (i) to determine the transport kinetics of AQP9 for various solutes. We will perform stopped-flow measurements on AQP9 proteoliposomes to characterize the transport kinetics for various solutes, including water, glycerol and larger solutes. The results will determine the physiological relevance of the AQP9-mediated transport of these solutes. (ii) to solve the structure of AQP9. We have already produced very well ordered two-dimensional (2D) crystals of AQP9 that diffract to about 3.8 ¿ resolution. We will continue to pursue electron crystallography of 2D crystals, but also x-ray crystallography of 3D crystals, to produce an atomic model of AQP9. (iii) to determine the transport kinetics of UreI, UT-Apl and UT-Ec for urea and water. We will perform stopped-flow measurements on proteoliposomes containing these urea channels to characterize their transport kinetics. The results will reveal similarities and differences in the function of these proteins. (iv) to obtain structural information on UreI, UT-Apl and UT-Ec. We will use biochemical and electron microscopic techniques to determine the oligomeric state of these urea channels. Our ultimate goal is to produce crystals (2D or 3D) of these proteins that will be suitable for structure determination by electron or x-ray crystallography. Relevance AQP9-mediated glycerol transport out of adipocytes and into the liver may be important to support gluconeogenesis in the fasted state. AQP9 is also permeated by arsenite and might contribute to the toxicity of arsenic ingestion. AQP9 may thus be a target for treating pathophysiological conditions resulting from eating disorders and arsenic poisoning. The availability of a structure for a UT might aid the development of novel diuretic compounds that selectively block urea reabsorption without interfering with the salt balance. UTs also play a crucial role in the survival of human pathogens. An atomic structure of the UT-Apl could thus potentially be used to develop specific inhibitors of bacterial urea transport. Transporters of the UAC family could be particularly potent targets for new antibiotics, since they do not have any homologs in eukaryotes.

摘要 尿素是哺乳动物体内的主要分解代谢产物，也是许多动物的重要氮源。 微生物。这项建议侧重于膜的结构和功能研究。 促进尿素跨膜转运的蛋白质，特别是 水通道蛋白(AQP)、尿素转运蛋白(UT)和尿素/酰胺通道(UAC)家族。我们 正在研究AQP9，它是所有已知的AQP中具有最广泛底物特异性的， 幽门螺杆菌Uac家族成员UreI和尿素转运蛋白 胸膜肺炎放线杆菌的UT-APL和致尿性大肠杆菌的UT-EC 菌株536。这项建议的具体目的是：(一)确定交通工具 AQP9在不同溶质中的动力学我们将进行停流测量 AQP9蛋白脂质体用于表征各种溶质的转运动力学， 包括水、甘油和更大的溶质。结果将决定生理学上的 AQP9介导的这些溶质运输的相关性。(二)着力破解结构性难题 AQP9。我们已经生产出了非常有序的二维(2D)晶体 AQP9衍射率约为3.8？分辨率。我们将继续追求电子 2D晶体的结晶学，也是3D晶体的X射线结晶学，以生产 AQP9的原子模型。(Iii)测定UreI、UT-APL的转运动力学 尿素和水的UT-EC。我们将进行停流测量 含有这些尿素通道的蛋白质脂质体来表征它们的转运动力学。 结果将揭示这些蛋白质在功能上的相似和不同。(四) 获取UreI、UT-APL和UT-EC的结构信息。我们将使用生化技术 和电子显微镜技术来确定这些尿素的低聚状态 频道。我们的最终目标是生产这些蛋白质的晶体(2D或3D)， 适用于电子或X射线结晶学结构测定。相关性 AQP9介导的甘油从脂肪细胞到肝脏的转运可能是重要的 以支持禁食状态下的糖异生。AQP9也被亚砷酸盐和 可能与砷摄入的毒性有关。因此，AQP9可能成为 治疗饮食失调和砷引起的病理生理状况 下毒了。UT结构的可用性可能有助于小说的发展 利尿剂化合物，选择性地阻止尿素的重吸收，而不干扰 盐分平衡。UTS在人类病原体的生存中也起着至关重要的作用。一颗原子弹 因此，UT-APL的结构可能被用来开发特定的抑制剂 细菌尿素运输。UAC家族的转运蛋白可能特别强大 新抗生素的目标，因为它们在真核生物中没有任何同系物。