Investigating the Mechanism of Activation of EphA4

项目摘要/摘要 多种治疗增生性和神经退行性疾病的方法，如癌症和肌萎缩侧索硬化 硬化症(ALS)，旨在干扰或抑制下游信号通路组件，如MAPK级联。 在针对受体酪氨酸激酶(RTK)的治疗开发中的一个关键障碍是经常 信号通路是相互连接的，这可能会导致偏离靶点的副作用。缓解这些问题的一种方法 挑战在于确定主动发挥作用的膜受体的激活机制 细胞功能异常。RTK，如促红细胞生成素(EPH)受体4，一直是 特别感兴趣的是潜在的毒品目标。然而，关于EphA4的结构信息缺乏， 提供对受体激活的洞察。在这项工作中，我们的目标是确定在 二聚体EphA4的激活以及研究功能缺失EphA4突变体的活性 互补的基于荧光和结构的研究，以验证我们的发现。在目标一中，我们建议 用冷冻电子显微镜阐明导致野生型EphA4激活的关键结构差异 (冷冻-EM)。在目标2中，我们建议表征已知的EphA4癌的异常活性和寡聚化 通过基于荧光的研究、生物物理和生物化学分析来筛选突变体。这些目标的结果将是 有助于我们对RTK的基本理解，并将有助于开发EphA4 增生性和神经退行性疾病的基于结构的药物设计。

Project Summary/Abstract Many therapeutics for proliferative and neurodegenerative disorders, such as cancer and amyotrophic lateral sclerosis (ALS), aim to disrupt or inhibit downstream signaling pathways components such as the MAPK cascade. A critical barrier in therapeutic development against Receptor Tyrosine Kinases (RTKs) is that oftentimes signaling pathways are interconnected, and this may lead to off-target side-effects. One method to mitigate these challenges is to determine the mechanism of activation of membrane receptors that actively play a role in abnormal cellular functions. RTKs, such as Erythropoietin-producing hepatoma (Eph) receptor 4, have been of particular interest as potential drug targets. However, there is a lack of structural information on EphA4 that provides insight into receptor activation. In this work, we aim to determine key structural differences during activation of dimeric EphA4 as well investigate the activity of loss-of-function EphA4 mutants using complementary fluorescence-based and structural studies to validate our findings. In aim one, we propose to elucidate critical structural differences resulting in wild type EphA4 activation using cryo-electron microscopy (cryo-EM). In aim 2, we propose to characterize aberrant activity and oligomerization of known EphA4 cancer mutants through fluorescence-based studies, biophysical, and biochemical assays. The results of these aims will contribute significantly to our underlying understanding of RTKs and will aid in efforts to develop EphA4 structure-based drug designs for proliferative and neurodegenerative disorders.