Structural and functional studies of urea channels

尿素通道的结构和功能研究

• 批准号: 7762749
• 负责人: THOMAS WALZ
• 金额: $ 21.77万
• 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/7762749
• 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