Structural and functional studies of urea channels

尿素通道的结构和功能研究

• 批准号: 7555922
• 负责人: THOMAS WALZ
• 金额: $ 21.96万
• 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/7555922
• 关键词: AQP9 gene; Actinobacillus pleuropneumoniae; Address; Adipocytes; Amides; Antibiotics; Arsenic; Arsenic Poisoning; Arsenites; Biochemical; Biological; Boston; Cells; Characteristics; Clinical; Collaborations; Crystallization; Crystallography; DNA; Data; Data Set; Development; Diuretics; Drug Design; E coli GlpF protein; Eating Disorders; Electron Microscopy; Electrons; Escherichia coli; Eukaryota; 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; 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，也是许多动物的重要氮源。 该提案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。 正在研究 AQP9，它在所有已知的 AQP 中具有最广泛的底物特异性， 来自幽门螺杆菌（UAC 家族成员）的 UreI 和尿素转运蛋白 来自胸膜肺炎放线杆菌的 UT-Apl 和来自尿路致病性大肠杆菌的 UT-Ec 菌株 536。该提案的具体目标是： (i) 确定运输 我们将对各种溶质的 AQP9 动力学进行停流测量。 AQP9 蛋白脂质体用于表征各种溶质的转运动力学， 包括水、甘油和较大的溶质，结果将决定生理学。 AQP9 介导的这些溶质运输的相关性 (ii) 来解决结构问题。 我们已经生产出了非常有序的二维（2D）晶体。 AQP9 衍射约为 3.8 ¿我们将继续追求电子化。 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、UT-Apl 的传输动力学。 我们将对尿素和水进行停流测量。 含有这些尿素通道的蛋白脂质体来表征它们的运输动力学。 结果将揭示这些蛋白质功能的相似点和差异 (iv)。 为了获得 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低聚状态 我们的最终目标是生产这些蛋白质的晶体（2D 或 3D）。 适用于通过电子或 X 射线晶体学进行结构测定。 AQP9 介导的甘油从脂肪细胞转运至肝脏可能很重要 在禁食状态下支持糖异生 AQP9 也被亚砷酸盐和渗透。 因此，AQP9 可能是 治疗由饮食失调和砷引起的病理生理状况 UT 结构的可用性可能有助于新颖的开发。 选择性阻断尿素重吸收而不干扰尿素的利尿化合物 盐平衡对于人类病原体的生存也起着至关重要的作用。 因此，UT-Apl 的结构可潜在地用于开发特定的抑制剂 UAC家族的细菌转运蛋白可能特别有效。 新抗生素的靶点，因为它们在真核生物中没有任何同源物。