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。为了进行反复信号，必须在释放后立即从突触中删除神经递质。在正常的大脑中，谷氨酸被谷氨酸转运蛋白（称为兴奋性氨基酸转运蛋白（EAATS））有效去除。这些分子在神经元和其他脑细胞的表面上发现。但是，在异常状态下，大量的谷氨酸可能导致接收神经细胞过度兴奋，从而导致损伤或死亡。该过程称为谷氨酸兴奋性，被认为是在脑缺血和头部创伤中看到的神经元丧失，并参与神经退行性疾病，例如亨廷顿和阿尔茨海默氏病。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是治疗性的。在某些条件下，EAAT的拮抗剂和激动剂都可以是神经保护作用，因此这种化合物的发展具有重要的生物医学重要性。由于缺乏EAAT3的重组表达系统，有效抑制剂的发展受到阻碍。当前测定EAAT的转运蛋白活性的方法很麻烦，并且缺乏哺乳动物EAAT3的高分辨率结构信息使理性抑制剂设计具有挑战性。该项目的目的是开发一种昆虫细胞表达系统，以产生纯活跃的人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，用于高分辨率X射线结构测定。将基于EAAT3的位置突变体创建一种新型的基于荧光的测定法，以测量微板格式的结合活性，以筛选蒙大拿大学合成的谷氨酸转运蛋白抑制剂。将采用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EAAT3转运活性的机制，并促进化合物的设计以阻断谷氨酸兴奋性兴奋性。公共卫生相关性：在中风，头部创伤，亨廷顿和阿尔茨海默氏病的疾病中，谷氨酸水平过高，导致神经元的永久丧失。在正常的大脑中，谷氨酸水平被谷氨酸转运蛋白（称为兴奋性氨基酸转运蛋白（EAATS））保持低。这项研究将有助于解释这些蛋白质如何起作用，并可能有助于开发新药，从而有助于防止高谷氨酸的影响。