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

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。为了使反复的信号发生，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蛋白（EAAT）的谷氨酸转运蛋白有效地清除。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性毒性，被认为有助于脑缺血和头部创伤中观察到的神经元损失，并涉及神经退行性疾病，如亨廷顿病和阿尔茨海默病。因此，旨在使谷氨酸转运蛋白恢复到正常表达和功能水平的治疗可能是治疗性的。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，因此开发此类化合物具有重要的生物医学意义。由于缺乏EAAT 3的重组表达系统，有效抑制剂的开发受到阻碍。目前检测EAAT转运蛋白活性的方法很麻烦，并且缺乏哺乳动物EAAT 3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EAAT 3作为毫克量的稳定的洗涤剂复合物。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。一种新的基于荧光的测定将创建，基于EAAT 3的定点突变体，以测量结合活性的微孔板格式筛选谷氨酸转运蛋白抑制剂在蒙大拿大学合成。将采取各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，并促进阻断谷氨酸兴奋毒性的化合物的设计。

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.