The Role of TDP-43 Phosphorylation in Protein Function and Neurodegeneration

描述（由申请人提供）：本项目的总体目标是确定调节TAR DNA结合蛋白（TDP-43）功能的因子。TDP-43是肌萎缩侧索硬化（ALS）和前颞叶变性（FTLD）（两种主要形式的神经变性）中细胞质包涵体中积累的主要疾病蛋白。TDP-43主要是核和介导RNA加工。TDP-43聚集伴随着核定位的丧失，这表明缺乏至少一种蛋白质的核功能与神经变性相关。此外，在家族性和散发性ALS-FTLD中发现的40多种显性突变突出了TDP-43在疾病中的重要性。然而，TDP-43在疾病中的作用仍然未知。关于调节TDP-43的因素以及翻译后修饰（PTM）是否影响TDP-43功能的信息很少。我们的初步搜索确定了特定的TDP- 43残基，在生理条件下进行磷酸化和多个其他预测的磷酸化位点。基于它们的位置，报告和预测的磷酸化位点可能会深刻影响TDP-43的关键过程，例如细胞运输和RNA相互作用。此外，几乎一半的患者相关TDP-43突变可能会增加蛋白磷酸化。我们假设TDP-43受磷酸化调节，并且几种疾病相关突变改变了磷酸化谱，对蛋白质功能产生有害影响。我们建议（目的1）测试磷酸化对蛋白质功能的影响，包括通过我们提出的分析鉴定的新磷酸化位点;（目的2）确定蛋白质活性和溶解度是否受到改变磷酸化谱的患者相关突变的影响;（目的3）表征TDP-43磷酸化在应激颗粒定位中的功能。细胞培养模型将用于通过质谱法鉴定磷酸盐。将在完善的细胞测定中使用定点突变体来探测磷酸化对蛋白质功能的作用，以监测TDP-43活性和溶解度。目前缺乏关于TDP-43生理磷酸化和与患者衍生突变体相关的信息，这强调了我们研究了解蛋白质功能及其在疾病中作用的重要性。我们的长期目标是确定调节TDP-43的因子和细胞通路，并有助于阐明导致神经退行性变的过程。 候选人长期以来一直对介导RNA加工的蛋白质的表征感兴趣，特别是对TDP-43功能的研究。NINDS促进神经科学研究多样性的教师发展奖将提供结构化培训，并专注于神经疾病机制。作为一个新成立的独立学院在爱德华A。在圣刘易斯大学生物化学和分子生物学Doisy部门，候选人致力于继续阐明与神经退行性疾病相关的分子机制。来自圣路易斯华盛顿大学生物化学和分子生物学系以及神经疾病希望中心的优秀导师团队将监测和评估研究进展和候选人职业目标的实现情况。作为职业发展计划的一部分，候选人将积极参加圣路易斯华盛顿大学的神经疾病希望中心和蛋白质聚集和神经变性希望中心计划。这将大大有助于候选人在神经退行性疾病领域的准备，并增加候选人研究的转化潜力。

DESCRIPTION (provided by applicant): The overall goal of this project is to identify factors that regulate the function of the TAR DNA binding protein (TDP-43). TDP-43 is the main disease protein accumulating in cytoplasmic inclusion bodies in amyotrophic lateral sclerosis (ALS) and front temporal lobar degeneration (FTLD), two major forms of neurodegeneration. TDP-43 is predominantly nuclear and mediates RNA processing. TDP-43 aggregation is accompanied by the loss of nuclear localization, suggesting that lack of at least one of the protein's nuclear functions is associated with neurodegeneration. In addition, the importance of TDP-43 in disease is highlighted by more than 40 dominant mutations found in familial and sporadic forms of ALS-FTLD. The role of TDP-43 in disease, however, remains unknown. Little information exists on factors that regulate TDP-43 and whether posttranslational modifications (PTMs) affect TDP-43 function. Our preliminary search identified specific TDP- 43 residues that undergo phosphorylation under physiological conditions and multiple other predicted phosphosites. Based on their positions, the reported and predicted phosphosites may profoundly affect key TDP-43 processes, e.g. cellular trafficking and RNA interactions. In addition, almost half of the patient-linked TDP-43 mutations potentially increase protein phosphorylation. We hypothesize that TDP-43 is regulated by phosphorylation and that several disease-associated mutations change the phosphorylation profile with deleterious consequences on protein function. We propose to (aim 1) test the effect of phosphorylation on protein function, including the novel phosphosites identified through our proposed analyses; (aim 2) determine if protein activity and solubility are affected by the patient-linked mutations that change the phosphorylation profile; and (aim 3) characterize the function of TDP-43 phosphorylation in stress granule localization. Cell culture models will be used to identify phosphosites by mass spectrometry. The role of phosphorylation on protein function will be probed using site- directed mutants in well-established cellular assays to monitor TDP-43 activity and solubility. The current lack of information regarding TDP-43 physiological phosphorylation and that associated with patient-derived mutants underscores the importance of our studies to understand protein function and its role in disease. Our long-term goal is to identify the factors and cellular pathways that regulate TDP-43 and to contribute in the elucidation of processes that cause neurodegeneration. The candidate has a long-standing interest in the characterization of proteins that mediate RNA processing, particularly the investigation of TDP-43 function. The NINDS Faculty Development Award to Promote Diversity in Neuroscience Research will provide structured training and focus on neurological disease mechanisms. As a recently established independent faculty in the Edward A. Doisy Department of Biochemistry and Molecular Biology at Saint Louis University, the candidate is committed to continuing the elucidation of molecular mechanisms associated with neurodegeneration. An outstanding team of mentors from the Department of Biochemistry and Molecular Biology and the Hope Center for Neurological Disorders at Washington University, St. Louis, will monitor and evaluate the research progress and the achievement of the candidate's career goals. As part of the career development program, the candidate will actively participate in the Hope Center for Neurological Disorders and the Hope Center Program on Protein Aggregation & Neurodegeneration at Washington University, St. Louis. This will greatly contribute to the candidate's preparation in the field of neurodegeneration and increase the translational potential of the candidate's research.