Determination of the mechanism underlying Thorase modulation of AMPARs mediated neurotransmission

项目总结/摘要 我的科学训练包括蛋白质生物化学在我的博士学位和博士后培训， 神经科学我的长期职业目标是成为一名专注于机制的独立调查员 与AAA+ ATP酶Thorase相关的神经系统疾病。我未来三年的目标是 获得结构生物学的额外知识和专业知识，以阐明Thorase是如何 在低聚复合物中结构化和组织化，与α-氨基-3-羟基-5-甲基-4- 异恶唑丙酸受体（AMPAR）。在一群杰出导师的帮助下，我们 开发了一个结构化的培训计划，其中包括广泛的动手研究培训蛋白质 结晶，负染色，和单颗粒冷冻电子显微镜，以及采取正式 课程，在研讨会上演讲，参加科学会议，并在赠款写作培训。本次培训 该计划将帮助我实现本K 01中提出的目标，并最终使我能够胜任 为R 01融资。Thorase在控制突触可塑性、学习和神经功能方面起着关键作用。 通过调节表面AMPAR的表达来增强记忆。Thorase的突变与 在某些情况下，表达Thorase功能丧失的新生儿表现出极端的 张力过高、脑病、癫痫和早逝。Thorase突变的小鼠在以下方面存在缺陷： 神经元信号和行为缺陷。与Thorase功能缺陷相关的异常可以 在患者和小鼠中，AMPAR拮抗剂perampanel使其正常化。这些发现表明 Thorase可能是神经系统疾病（如精神分裂症）的重要介质， AMPAR介导的多巴胺能神经传递受损Thorase如何调节AMPAR- 介导的多巴胺能神经传递仍有待阐明。已知AAA+ ATP酶可形成 低聚复合物对于它们的功能至关重要。Thorase的结构和确切的数量 其寡聚复合物中存在的原聚体目前是未知的。为了探究这些问题，我的导师们 我设计了一系列的研究，这些研究将提供重要的信息， Thorase介导的AMPAR运输/再循环，以及神经元中的其他Thorase功能。实现 这些目标，该提案的研究目的是（i）确定不同的低聚状态， Thorase使用单颗粒冷冻EM;（ii）确定对AMPAR至关重要的Thorase的特定残基 （iii）确定与AMPAR亚基的C-末端结合的Thorase的晶体结构 GluA 2;和（iv）鉴定Thorase寡聚体的新的相互作用蛋白伴侣 配合物了解Thorase的结构及其寡聚复合物如何与AMPAR相互作用， 推进我们对Thorase如何通过AMPAR信号传导介导突触可塑性变化的理解。 这项研究还可能为Thorase和AMPAR介导的细胞凋亡识别新的潜在治疗靶点。 神经系统疾病

Project Summary/Abstract My scientific training includes protein biochemistry during my doctorate and post-doctoral training in neuroscience. My long-term career goal is to become an independent investigator focusing on mechanisms of neurologic diseases related to the AAA+ ATPase Thorase. My goal over the next three years is to acquire additional knowledge and expertise in structural biology in order to elucidate how Thorase is structured and organized in oligomeric complexes to interact with α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors (AMPARs). With the help of a team of outstanding mentors, we have developed a structured training program that includes extensive hands-on research training in protein crystallization, negative staining, and single particle cryo-electron microscopy, as well as taking formal courses, presenting at seminars, attending scientific meetings, and training in grant writing. This training program will help me achieve the goals proposed in this K01 and ultimately prepare me to be in a position to compete for R01 funding. Thorase plays a critical role in controlling synaptic plasticity, learning, and memory by modulating the expression of surface AMPARs. Mutations in Thorase have been associated with schizophrenia and in some cases, neonates expressing loss-of-function Thorase demonstrate extreme hypertonia, encephalopathy, seizures, and early death. Mice with Thorase mutations have defects in glutamatergic signaling and deficits in behavior. The abnormalities linked to defects in Thorase function can be normalized by the AMPAR antagonist, perampanel, both in patients and mice. These findings suggest that Thorase could be an important mediator of neurologic diseases, such as schizophrenia, that are linked to compromised AMPAR-mediated glutamatergic neurotransmission. How Thorase modulates AMPAR- mediated glutamatergic neurotransmission remains to be elucidated. AAA+ ATPases are known to form oligomeric complexes that are critical for their functions. The structure of Thorase and the exact number of protomers present in its oligomeric complex are currently unknown. To probe these questions, my mentors and I have designed a series of studies that will provide vital information underlying the mechanism of Thorase-mediated AMPAR trafficking/recycling, as well as other Thorase functions in neurons. To attain these goals, the research aims of the proposal are to (i) determine the different oligomeric states of Thorase using single particle cryo-EM; (ii) identify specific residues of Thorase that are critical for AMPAR trafficking; (iii) determine the crystal structure of Thorase bound to the C-terminus of the AMPAR subunit GluA2 using x-ray crystallography; and (iv) identify new interacting protein partners of Thorase oligomeric complexes. Knowing the structure of Thorase and how its oligomeric complex interacts with AMPARs will advance our understanding of how Thorase mediates changes in synaptic plasticity via AMPAR signaling. This study may also identify new potential therapeutic targets for Thorase and AMPAR-mediated neurologic disease.