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

基本信息

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
项目类别：
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&apos 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

项目摘要

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（由申请人提供）：谷氨酸由突触前神经元释放并刺激突触后神经元中的电信号。为了发生重复的信号传导，神经递质必须在释放后立即从突触中去除。在正常大脑中，谷氨酸被谷氨酸转运蛋白（称为兴奋性氨基酸转运蛋白（EAAT））有效去除。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，大量的谷氨酸会导致接收神经细胞过度兴奋，从而导致损伤或死亡。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损失，并与亨廷顿病和阿尔茨海默氏病等神经退行性疾病有关。因此，旨在使谷氨酸转运蛋白恢复正常表达和功能水平的治疗可能具有治疗作用。 EAAT 的拮抗剂和激动剂在某些条件下都可以具有神经保护作用，因此此类化合物的开发具有重要的生物医学重要性。由于缺乏 EAAT3 重组表达系统，有效抑制剂的开发受到阻碍。目前测定 EAAT 转运蛋白活性的方法很麻烦，而且缺乏哺乳动物 EAAT3 的高分辨率结构信息，使得合理的抑制剂设计具有挑战性。该项目的目标是开发一种昆虫细胞表达系统，以生产纯的、活性的人 EAAT3，作为毫克量的稳定洗涤剂复合物。纯化的蛋白质将用于配体筛选、生物物理表征和结晶，以进行高分辨率 X 射线结构测定。将创建一种基于 EAAT3 定点突变体的新型荧光测定法，以测量微孔板形式的结合活性，以筛选蒙大拿大学合成的谷氨酸转运蛋白抑制剂。将采取多种实验方法来生产和优化适合高分辨率结构分析的EAAT3晶体，以阐明人类EAAT3转运活性的机制并促进设计阻断谷氨酸兴奋毒性的化合物。公共健康相关性：在中风、头部外伤、亨廷顿舞蹈症和阿尔茨海默病中，谷氨酸水平过高，导致神经元永久性丧失。在正常大脑中，谷氨酸转运蛋白（称为兴奋性氨基酸转运蛋白（EAAT））将谷氨酸水平保持在较低水平。这项研究将有助于解释这些蛋白质的工作原理，并可能有助于开发有助于预防高谷氨酸盐影响的新药物。