Mechanisms and Binding-Sites in Adenylate Kinase-Dependent CFTR Gating

腺苷酸激酶依赖性 CFTR 门控的机制和结合位点

• 批准号: 8517797
• 负责人: Christoph Oskar Randak
• 金额: $ 12.74万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-13 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517797
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Amino Acids; Arts; Binding; Binding Sites; Biochemical; Biological; Carrier Proteins; Chloride Channels; Chloride Ion; Chlorides; Collaborations; Coupled; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Dependency; Development; Diarrhea; Diphosphates; Disease; Educational process of instructing; Electrophysiology (science); Elements; Environment; Family; Funding; Genes; Goals; Heart; Hydrolysis; Instruction; Iowa; Knowledge; Label; Laboratories; Laboratory Research; Length; Membrane; Membrane Proteins; Mentors; Methods; Molecular; Mutation; Nucleotides; Outcome; Physicians; Physiological; Principal Investigator; Protein Biochemistry; Protein Chemistry; Protein Family; Proteins; Publishing; Quality of life; Recombinants; Research; Research Personnel; Research Proposals; Role; Scientist; Site; Structure-Activity Relationship; Techniques; Testing; Training; Universities; Work; Writing; abstracting; adenylate kinase; base; career; career development; cystic fibrosis patients; experience; human disease; improved; in vivo; inhibitor/antagonist; innovation; inorganic phosphate; kinase inhibitor; member; membrane activity; novel; patch clamp; pediatric department; prevent; programs; protein function; reconstitution; skills; success; teacher; tripolyphosphate

DESCRIPTION (provided by applicant): This application proposes a career development program for Dr. Christoph O. Randak to develop into an independent physician-scientist and academic teacher and mentor. The heart of the proposal is an intensive laboratory training experience to develop scientific skills and understanding in protein chemistry and biochemistry of membrane proteins. The cystic fibrosis transmembrane conductance regulator (CFTR), a regulated chloride channel, is a representative of one of the largest families of membrane proteins, the adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters. Mutations in the gene encoding CFTR cause cystic fibrosis (CF). Understanding the molecular mechanisms of CFTR channel gating will facilitate the development of means to cure and control CF. Many previous studies have focused on CFTR's ATPase activity and how it governs CFTR gating. Recent work has shown that CFTR also interacts with adenosine 5'-monophosphate (AMP) and that adenylate kinase activity (ATP:AMP phosphotransfer) can gate CFTR. The objective of this research proposal is to identify key mechanisms and key amino acid residues involved in AMP-binding and adenylate kinase activity of CFTR. Using labeling techniques with azido-nucleotides and CFTR patch-clamping, the hypothesis will be tested that AMP and ATP interact cooperatively with CFTR, and that AMP prevents ATP hydrolysis and induces ATP:AMP phosphotr-ansfer. The results are expected to elucidate how ATP and AMP mutually influence their interaction with CFTR, and how AMP influences CFTR's enzymatic activity; it is also expected to identify key residues involved in AMP-binding to CFTR. The program will provide Dr. Randak with formal career mentoring and guidance. Dr. Michael J. Welsh will assume responsibility as mentor and Drs. Jeffrey C. Murray, Paul B. McCray Jr., and Kevin P. Campbell will be advisors. Additional objectives are to increase Dr. Randak's teaching and mentoring experience, to guide him in establishing a research laboratory, to increase his skills in scientific speaking and writing, to help him expand his contacts and collaborations with other scientists, and to prepare him to apply for independent research funding. The University of Iowa will provide a state-of-art research environment dedicated to Dr. Randak's scientific development and academic success. RELEVANCE (See instructions): Knowledge from these studies will provide a framework to develop activators and inhibitors of CFTR channel function. Such compounds may be useful for the treatment of diseases associated with too little (cystic fibrosis) or too much (secretory diarrhea) CFTR chloride currents. Because CFTR is an ABC transporter, this knowledge may also impact treatments of other ABC transporter-related diseases. (End of Abstract)

描述（由申请人提供）：本申请为Christoph O. Randak博士提供了一项职业发展计划，以发展为独立的医师科学家，学术老师和导师。该提案的核心是一种强化的实验室培训经验，旨在发展蛋白质化学和膜蛋白质生物化学的科学技能和理解。囊性纤维化跨膜电导调节剂（CFTR）是一种受调节的氯化物通道，是最大的膜蛋白家族之一的代表，即5'-三磷酸腺苷（ATP） - 结合卡塞特（ABC）转运蛋白。编码CFTR的基因中的突变引起囊性纤维化（CF）。了解CFTR通道门控的分子机制将有助于治愈和控制CF的方法的发展。许多先前的研究都集中在CFTR的ATPase活动及其如何控制CFTR门控。最近的工作表明，CFTR还与腺苷5'-单磷酸腺苷相互作用（AMP），并且腺苷酸激酶活性（ATP：AMP磷酸转移）可以栅极CFTR。该研究建议的目的是确定参与CFTR的AMP结合和腺苷酸激酶活性的关键机制和关键氨基酸残基。使用偶氮核苷酸和CFTR斑块链接的标记技术，该假设将测试AMP和ATP与CFTR合作，并且AMP可以防止ATP水解和诱导ATP：AMP Phosphotr-phosphotr-andfer。预期结果将阐明ATP和AMP如何互惠互相影响其与CFTR的相互作用，以及AMP如何影响CFTR的酶活性。还可以预计将确定与CFTR的AMP结合有关的关键残基。该计划将为Randak博士提供正式的职业指导和指导。迈克尔·J·威尔士（Michael J. Welsh）博士将担任导师和博士的责任。 Jeffrey C. Murray，Paul B. McCray Jr.和Kevin P. Campbell将担任顾问。其他目标是提高兰德克博士的教学和指导经验，指导他建立研究实验室，以提高他在科学说话和写作方面的技能，以帮助他扩大与其他科学家的联系和合作，并为他申请独立研究资金做好准备。爱荷华大学将提供最先进的研究环境，该研究环境致力于兰德克博士的科学发展和学术成就。相关性（请参阅说明）：这些研究的知识将为开发CFTR通道功能的激活因子和抑制剂提供一个框架。这种化合物可能对治疗与太少（囊性纤维化）或过多（分泌性腹泻）CFTR氯化物电流相关的疾病有用。由于CFTR是ABC转运蛋白，因此这些知识也可能影响其他与ABC转运蛋白相关疾病的治疗方法。 （抽象的结尾）