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

描述(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的电信号。为了重现信号，神经递质必须在释放后不久从突触中移除。在正常大脑中，谷氨酸被称为兴奋性氨基酸转运体(EAATs)的谷氨酸转运体有效地清除。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的过度兴奋，导致损伤或死亡。这一过程被称为谷氨酸兴奋毒性，被认为是导致脑缺血和头部创伤中神经元丢失的原因之一，并与亨廷顿氏症和阿尔茨海默氏症等神经退行性疾病有关。因此，旨在使谷氨酸转运体恢复到正常表达和功能水平的治疗可能具有治疗作用。EAATs的拮抗剂和激动剂在一定条件下都具有神经保护作用，因此开发这类化合物具有重要的生物医学意义。由于缺乏EAAT3的重组表达系统，有效的抑制剂的开发受到阻碍。目前检测EAATs转运蛋白活性的方法比较繁琐，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，以生产纯的、有活性的人EAAT3作为一种稳定的毫克量的洗涤剂复合体。纯化的蛋白质将用于配基筛选、生物物理表征和高分辨X射线结构测定的结晶。基于EAAT3的定点突变体，将创建一种新的基于荧光的分析方法，以微板形式测量结合活性，用于筛选蒙大拿大学合成的谷氨酸转运蛋白抑制剂。为了阐明人类EAAT3转运活性的机制，便于设计阻断谷氨酸兴奋性毒性的化合物，人们将采取各种实验方法来制备和优化适合于高分辨结构分析的EAAT3晶体。 与公共健康相关：在中风、头部创伤、亨廷顿病和阿尔茨海默病中，谷氨酸水平变得过高，导致神经元永久性丧失。在正常大脑中，谷氨酸转运体被称为兴奋性氨基酸转运体(EAATs)，使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是如何发挥作用的，并可能有助于开发有助于防止高谷氨酸影响的新药。

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.