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

突触核蛋白病中 14-3-3 磷酸化的原因和下游影响

• 批准号: 10606132
• 负责人: Frank Sanders Pair
• 金额: $ 4.13万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606132
• 关键词: Acceleration; Affect; Age; Apoptosis; Binding; Bioinformatics; Biological; Biological Assay; Brain; Cell Death; Cell physiology; Cells; Charge; Clinical Skills; Co-Immunoprecipitations; Cognitive; Communication; Data; Dementia with Lewy Bodies; Disease; Disease model; Dopaminergic Cell; Educational process of instructing; Event; Exposure to; Family; Future; Genetic; Goals; Head; Human; Immunoprecipitation; In Vitro; Incidence; Knock-in; Knock-in Mouse; Mass Spectrum Analysis; Mediating; Mentors; Mentorship; Modeling; Motor; Multiple System Atrophy; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotoxins; Parkinson Disease; Pathologic; Pathology; Pathway Analysis; Pathway interactions; Patient Care; Patients; Phosphorylation; Phosphotransferases; Physicians; Play; Population; Predisposition; Protein Family; Protein Isoforms; Proteins; Proteomics; Rattus; Risk; Role; Rotenone; Sampling; Scientist; Severities; Signal Transduction; Site; Solubility; Substantia nigra structure; Symptoms; Techniques; Testing; Toxic Environmental Substances; Toxic effect; Trichloroethylene; United States National Institutes of Health; Western Blotting; Work; alpha synuclein; attenuation; axon growth; bioinformatics tool; candidate selection; career development; cognitive function; disorder risk; experimental study; in vivo; inhibitor; insight; kinase inhibitor; knock-down; mimetics; mutant; new therapeutic target; novel; novel therapeutics; overexpression; pars compacta; pharmacologic; protective effect; protein folding; protein protein interaction; research and development; response; skills; synucleinopathy; therapeutic target; toxicant

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.

项目摘要/摘要 该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。 该提案的重点是理解突触核力病中14-3-3磷酸化的机制。 帕金森氏病（PD）和痴呆症具有路易尸体（DLB）是突触性的，其特征是其特征 α-核蛋白（αSyn）的聚集。这些疾病的特征是渐进电动机的混合 认知和自主症状。我们的实验室研究了14-3-3蛋白在这些疾病中扮演的角色。 14-3-3 是一个普遍表达的蛋白质家族，代表大脑中所有固体蛋白的几乎1％ 主要通过蛋白质 - 蛋白质相互作用（PPI）发挥其功能。我们在 尤其是14-3-3θ同工型，受到保护，不受α同步，而抑制这些蛋白质会导致增加 毒性。此外，我们发现PD和DLB患者的人体皮质裂解物增加了14-3-3θ 与健康，年龄匹配的对照相比，S232的磷酸化。测试14-3-3θ的影响 磷酸化，我们发现不可磷酸化的S232A突变体在PD模型中受到保护，而不是磷酸化的。 磷酸化S232D突变体没有保护或加速毒性。这些结果使我们进入 假设的S232磷酸化在14-3-3θ的保护函数中起作用。目标 该项目是为了了解S232磷酸化的原因和下游效应。在目标1 i 建议确定激酶引起这种磷酸化的建议。我将与正在的克里斯托弗·威利博士一起工作 UAB Kinome核心的电荷。我将同时使用候选方法和无偏见的方法来识别关键 通过PD相关毒物，三氯乙烯（TCE）和 烤面包酮。对于一种公正的方法，我将使用pamstation kinomics芯片来识别被激活的激酶 烤面包酮和TCE。我将使用激酶抑制剂验证这些结果，并评估生物学 通过遗传敲低以及激酶的药物抑制剂对鉴定激酶的相关性。博士 威利将指导我采用生物信息学技术以及选择候选激酶以进行进一步研究。 在AIM 2中，我想了解数百个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詹姆斯·莫布利（James Mobley）博士一起执行质量 来自WildType，S232A或S232D敲入皮质脑裂解物的共免疫沉淀的光谱法 了解蛋白质网络如何与14-3-3θ磷酸化的响应响应14-3-3θ变化。这 除了我的导师Talene Yacoubian博士以及我的委员会的意见外，实验还将帮助 我达到了对神经变性，发展实验室和生物信息学技能的不断理解的目标， 沟通技巧，教学和心理技能以及未来患者护理所需的临床技能。全部 这些将帮助我成为一个专注于神经退行性疾病的出色身体科学家。