Causes and Downstream Effects of 14-3-3 Phosphorylation in Synucleinopathies

项目总结/摘要 这个NIH F31应用程序描述了PI的指导研究和职业发展的四年计划。 该提案的重点是了解突触核蛋白病中14-3-3磷酸化的机制。 帕金森氏病（PD）和路易体痴呆（DLB）是突触核蛋白病，其特征在于它们的 α-突触核蛋白（αsyn）的聚集。这些疾病的特点是混合进行性运动， 认知和自主神经症状我们的实验室研究14-3-3蛋白在这些疾病中的作用。14-3-3s 是一个普遍表达的蛋白质家族，占大脑中所有可溶性蛋白质的近1%， 主要通过蛋白质-蛋白质相互作用（PPIs）发挥其功能。我们已经发现了14-3-3蛋白， 特别是14-3-3θ亚型，对αsyn具有保护作用，而抑制这些蛋白质会导致增加 毒性此外，我们发现PD和DLB患者的人皮质裂解物显示14-3-3θ增加， S232的磷酸化与健康的年龄匹配的对照相比。测试14-3-3θ的影响 我们发现，不可磷酸化的S232 A突变体在PD模型中具有保护作用，而 磷酸化模拟S232 D突变体没有表现出保护作用或加速毒性。这些结果使我们 假设S232磷酸化在14-3-3θ保护功能丧失中起作用。的目标 本项目旨在了解S232磷酸化的原因和下游效应。目标1 I 我们建议确定是什么激酶导致了这种磷酸化。我将与克里斯托弗·威利博士合作，他在 负责UAB Kinome Core。我将使用候选方法和无偏见的方法来确定关键 通过PD相关毒物三氯乙烯（TCE）和 鱼藤酮为了一个公正的方法，我将使用PamStation Kinomics芯片来识别由 鱼藤酮和三氯乙烯。我将利用激酶抑制剂在体外验证这些结果，并将评估生物学特性。 通过基因敲低鉴定的激酶的相关性以及激酶的药理学抑制剂。博士 Willey将指导我进行生物信息学技术以及选择候选激酶进行进一步研究。 在目标2中，我想了解数百种14-3-3 PPI的下游后果。我会 与UAB质谱和蛋白质组学核心负责人James Mobley博士合作， 来自野生型、S232 A或S232 D敲入皮质脑裂解物的共免疫沉淀物的光谱分析， 了解蛋白质网络如何结合到14 - 3 - 3θ改变响应14-3-3θ磷酸化。的 除了我的导师Talene Yacoubian博士和我的委员会的投入之外， 我达到了我的目标，增加对神经变性的理解，发展实验室和生物信息学技能， 沟通技巧，教学和指导技能，以及未来病人护理所需的临床技能。所有 这些将帮助我成为一名专注于神经退行性疾病的优秀医生兼科学家。

Project Summary/Abstract This NIH F31 application describes a four-year plan for mentored research and career development for the PI. This proposal is focused on understanding the mechanisms of 14-3-3 phosphorylation in synucleinopathies. Parkinson’s Disease (PD) and Dementia with Lewy bodies (DLB) are synucleinopathies characterized by their aggregation of alpha-synuclein (αsyn). These diseases are characterized by a mix of progressive motor, cognitive, and autonomic symptoms. Our lab studies the role 14-3-3 proteins play in these diseases. 14-3-3s are a ubiquitously expressed family of proteins representing nearly 1% of all soluble protein in the brain that mainly exert their functions through protein-protein interactions (PPIs). We have found 14-3-3 proteins, in particular the 14-3-3θ isoform, are protective against αsyn while inhibition of these proteins leads to increased toxicity. Furthermore, we found that human cortical lysates of PD and DLB patients showed increased 14-3-3θ phosphorylation at S232 compared to healthy, age-matched controls. Testing the impact of 14-3-3θ phosphorylation, we found that the non-phosphorylatable S232A mutant is protective in PD models, while the phosphomimetic S232D mutant showed no protection or accelerated toxicity. These results lead us to hypothesize S232 phosphorylation plays a role in 14-3-3θ’s loss of protective functions. The goal of this project is to understand the causes and downstream effects of S232 phosphorylation. In Aim 1 I propose to identify what kinases cause this phosphorylation. I will work with Dr. Christopher Willey, who is in charge of the UAB Kinome Core. I will use both a candidate approach and non-biased approach to identify key kinases that increase 14-3-3 phosphorylation by PD-associated toxicants, trichloroethylene (TCE) and rotenone. For an unbiased approach, I will use the PamStation Kinomics chip to identify kinases activated by rotenone and TCE. I will validate these results in-vitro utilizing kinase inhibitors and will evaluate the biological relevance of identified kinases by genetic knockdown as well as pharmacological inhibitors of the kinases. Dr. Willey will guide me on the bioinformatic techniques as well as selection of candidate kinases for further study. In Aim 2 I want to understand what the downstream consequences are for the hundreds of 14-3-3 PPIs. I will work with Dr. James Mobley, head of UAB’s Mass spectrometry and proteomics core, to perform mass spectrometry of co-immunoprecipitates from wildtype, S232A or S232D knock-in cortical brain lysates to understand how protein networks binding to 14-3-3θ change in response to 14-3-3θ phosphorylation. The experiments in addition to the input from my mentor, Dr. Talene Yacoubian as well as my committee will help me meet my goals of increasing understanding of neurodegeneration, develop lab and bioinformatic skills, communication skills, teaching and mentorship skills, and clinical skills necessary for future patient care. All these will help me become an excellent physician-scientist focused on neurodegenerative disorders.