Structural and functional studies of urea channels

尿素通道的结构和功能研究

• 批准号: 7351221
• 负责人: THOMAS WALZ
• 金额: $ 27.02万
• 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 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7351221
• 关键词: AQP9 gene; Actinobacillus pleuropneumoniae; Address; Adipocytes; Amides; Antibiotics; Arsenic; Arsenic Poisoning; Arsenites; Biochemical; Biological; Boston; Cells; Characteristics; Class; Clinical; Collaborations; Condition; Crystallization; Crystallography; DNA; Data; Data Set; Development; Diuretics; Drug Design; E coli GlpF protein; Eating Disorders; Electron Microscopy; Electrons; Escherichia coli; Eukaryota; Eukaryotic Cell; Family; Fasting; Future; Genomics; 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; Nitrogen; Numbers; Pattern; Phase; Physiological; Play; Proteins; Rattus; Recombinant Proteins; Recombinants; 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; 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.

抽象的 尿素是哺乳动物的主要分解代谢物，也是许多许多人的氮源 微生物。该提案的重点是膜的结构和功能研究 促进跨膜尿素运输的蛋白质，特别是成员 Aquaporin（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，该AQP9在所有已知的AQP中具有最广泛的底物特异性， UREI来自幽门螺杆菌，UAC家族的成员和尿素转运蛋白 来自肌动杆菌的UT-APL尿液发育大肠杆菌和UT-EC的UT-APL 菌株536。该提案的具体目的是：（i）确定运输 AQP9的动力学用于各种解决方案。我们将对停止流量进行测量 AQP9蛋白脂质体以表征各种溶剂的传输动力学， 包括水，甘油和较大的溶剂。结果将决定生理 AQP9介导的这些解决方案的转运的相关性。 （ii）解决结构 AQP9。我们已经产生了非常有序的二维（2D）晶体 AQP9衍射至约3.8€。我们将继续追求电子 2D晶体的晶体学，以及3D晶体的X射线晶体学，以产生 AQP9的原子模型。 （iii）确定UREI的运输动力学，UT-APL 和UT-EC用于尿素和水。我们将对停止流量进行测量 含有这些尿素通道的蛋白质脂质体以表征其运输动力学。 结果将揭示这些蛋白质功能的相似性和差异。 （iv） 获取有关UREI，UT-APL和UT-EC的结构信息。我们将使用生化 和电子显微镜技术来确定这些尿素的寡聚状态 频道。我们的最终目标是生产这些蛋白质的晶体（2D或3D） 适合通过电子或X射线晶体学测定结构。关联 AQP9介导的甘油从脂肪细胞和肝脏传输可能很重要 支持禁食状态下的谷杆菌发生。 AQP9也被砷和 因此，AQP9可能是 治疗饮食失调和砷引起的病理生理状况 中毒。 UT的结构的可用性可能有助于开发新颖 利尿化合物，可有选择地阻断尿素重吸收而不会干扰 盐平衡。 UTS在人类病原体的生存中也起着至关重要的作用。原子 因此，UT-APL的结构可能可用于开发特定的抑制剂 细菌尿素运输。 UAC家族的运输商可能特别有效 新抗生素的靶标，因为它们在真核生物中没有任何同源物。