High-Level Expression of Human EAAT3 for Biochemical and Structural Analysis

用于生化和结构分析的人 EAAT3 高水平表达

• 批准号: 8035863
• 负责人: MICHAEL PATRICK KAVANAUGH
• 金额: $ 26.94万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035863
• 关键词: Adjuvant; Agonist; Alzheimer' s Disease; Amino Acid Sequence; Amino Acid Transporter; Archaea; Aspartate; Binding; Biochemical; Biological Assay; Brain; Cells; Cerebral Ischemia; Cessation of life; Complex; Craniocerebral Trauma; Crystallization; Cysteine; Cytoplasm; Detergents; Development; EAAT3; Excitatory Amino Acids; Fab Immunoglobulins; Fluorescence; Fluorescent Dyes; Fluorescent Probes; Glutamate Transporter; Glutamates; Glutamic Acid; Goals; Homeostasis; Human; Huntington Disease; In Vitro; Insecta; Integral Membrane Protein; Investigation; Ions; Kinetics; Lead; Life; Ligand Binding; Ligands; Liposomes; Mammals; Measurement; Measures; Membrane; Membrane Lipids; Methods; Modeling; Molecular; Molecular Conformation; Monoclonal Antibodies; Montana; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurons; Neurotransmitters; Performance; Pharmaceutical Preparations; Process; Property; Protein Isoforms; Proteins; Pyrococcus horikoshii; Radioactive; Recombinants; Recurrence; Reporter; Research; Resolution; Roentgen Rays; Screening procedure; Signal Transduction; Site; Stroke; Structural Models; Structure; Surface; Synapses; System; Therapeutic; Transmembrane Domain; Tryptophan; Universities; Ursidae Family; Work; base; brain cell; design; enzyme activity; excitotoxicity; inhibitor/antagonist; milligram; mutant; neuron loss; novel; postsynaptic; presynaptic; prevent; proteoliposomes; small molecule; therapeutic target; uptake

DESCRIPTION (provided by applicant): Glutamate is released by presynaptic neurons and stimulates an electrical signal in postsynaptic neurons. In order for recurrent signaling to occur, the neurotransmitter must be removed from the synapse soon after release. In the normal brain, glutamate is efficiently removed by glutamate transporters, known as excitatory amino acid transporters (EAATs). These molecules are found on the surface of neurons and other brain cells. In abnormal states, however, high amounts of glutamate can lead to overexcitation of the receiving nerve cell, resulting in damage or death. This process, known as glutamate excitotoxicity, is thought to contribute to neuronal loss seen in cerebral ischemia and head trauma, and is involved in neurodegenerative conditions such as Huntington's and Alzheimer's diseases. Therefore, treatments aimed at returning glutamate transporters to normal levels of expression and function may be therapeutic. Both antagonists and agonists of EAATs could be neuroprotective under certain conditions, and so development of such compounds is of significant biomedical importance. Development of effective inhibitors is impeded by lack of a recombinant expression system for EAAT3. Current methods to assay the transporter activity of EAATs are cumbersome, and the lack of high-resolution structural information for mammalian EAAT3 makes rational inhibitor design challenging. The goal of this project is to develop an insect cell expression system to produce pure, active human EAAT3 as a stable detergent complex in milligram quantities. The purified protein will be used for ligand screening, biophysical characterization and crystallization for high resolution X-ray structure determination. A novel fluorescence-based assay will be created, based on site-directed mutants of EAAT3, to measure binding activity in a microplate format for screening of glutamate transporter inhibitors synthesized at the University of Montana. A variety of experimental approaches will be taken to produce and optimize crystals of EAAT3 suitable for high-resolution structural analysis, in order to elucidate the mechanism of human EAAT3 transport activity and facilitate the design of compounds to block glutamate excitotoxicity. PUBLIC HEALTH RELEVANCE: In stroke, head trauma, Huntington's and Alzheimer's diseases, levels of glutamate become too high, causing the permanent loss of neurons. In the normal brain, glutamate levels are kept low by glutamate transporters, known as excitatory amino acid transporters (EAATs). This research will help explain how these proteins work and possibly aid in the development of new drugs that will help prevent the effects of high glutamate.

描述（申请人提供）：谷氨酸由突触前神经元释放，刺激突触后神经元的电信号。为了使信号循环发生，神经递质必须在释放后很快从突触中移除。在正常的大脑中，谷氨酸被谷氨酸转运体，即兴奋性氨基酸转运体（EAATs）有效地去除。这些分子存在于神经元和其他脑细胞的表面。然而，在异常状态下，大量谷氨酸可导致接收神经细胞过度兴奋，导致损伤或死亡。这一过程被称为谷氨酸兴奋性毒性，被认为会导致脑缺血和头部创伤中出现的神经元丧失，并与亨廷顿氏病和阿尔茨海默病等神经退行性疾病有关。因此，旨在使谷氨酸转运体的表达和功能恢复到正常水平的治疗可能是治疗性的。EAATs的拮抗剂和激动剂在某些条件下都具有神经保护作用，因此开发此类化合物具有重要的生物医学意义。缺乏EAAT3的重组表达系统阻碍了有效抑制剂的开发。目前测定EAATs转运体活性的方法很繁琐，而且缺乏哺乳动物EAAT3的高分辨率结构信息，这给合理的抑制剂设计带来了挑战。该项目的目标是开发一种昆虫细胞表达系统，以产生纯的、活性的人类EAAT3作为一种稳定的毫克级洗涤剂复合物。纯化后的蛋白将用于配体筛选、生物物理表征和高分辨率x射线结构测定的结晶。一种基于EAAT3位点定向突变体的新型荧光检测将被创建，以微孔板形式测量结合活性，用于筛选在蒙大拿大学合成的谷氨酸转运蛋白抑制剂。为了阐明人体EAAT3转运活性的机制，并为设计阻断谷氨酸兴奋毒性的化合物提供便利，我们将采用多种实验方法来制备和优化适合高分辨率结构分析的EAAT3晶体。