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

描述(由申请人提供)：该项目的总体目标是确定调节TAR DNA结合蛋白(TDP-43)功能的因素。TDP-43是肌萎缩侧索硬化症(ALS)和前颞叶变性(FTLD)这两种主要的神经退行性变的主要疾病蛋白，积聚在胞浆包涵体中。TDP-43主要是核的，参与RNA的加工。TDP-43的聚集伴随着核定位的丧失，这表明该蛋白至少有一项核功能的缺失与神经退化有关。此外，在家族性和散发性ALS-FTLD中发现的40多个显性突变突显了TDP-43在疾病中的重要性。然而，TDP-43在疾病中的作用仍不清楚。关于调控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在生理条件下发生磷酸化的特定TDP-43残基和多个其他预测的亚磷酸盐。根据它们的位置，报道和预测的亚磷酸盐可能会深刻影响关键的TDP-43过程，例如细胞运输和RNA相互作用。此外，几乎一半与患者相关的TDP-43突变可能增加蛋白质磷酸化。我们假设TDP-43受磷酸化调控，几个与疾病相关的突变改变了磷酸化谱，对蛋白质功能造成有害后果。我们建议(目的1)测试磷酸化对蛋白质功能的影响，包括通过我们建议的分析确定的新的亚磷酸位点；(目的2)确定患者相关突变是否影响蛋白质的活性和可溶性；以及(目的3)表征TDP-43磷酸化在应激颗粒定位中的功能。细胞培养模型将被用来通过质谱学来鉴定亚磷酸盐。磷酸化在蛋白质功能上的作用将使用成熟的细胞检测中的定点突变体来探索，以监测TDP-43的活性和溶解性。目前缺乏关于TDP-43生理磷酸化的信息以及与患者来源的突变相关的信息，这突显了我们研究了解蛋白质功能及其在疾病中的作用的重要性。我们的长期目标是确定调节TDP-43的因素和细胞通路，并在阐明导致神经退变的过程中做出贡献。这位候选人长期以来一直对介导RNA加工的蛋白质的特征，特别是对TDP-43功能的研究感兴趣。NINDS促进神经科学研究多样性的教师发展奖将提供有组织的培训，并专注于神经疾病机制。作为圣路易斯大学爱德华·A·多西生物化学和分子生物学系最近成立的独立教员，这位候选人致力于继续阐明与神经退行性变相关的分子机制。来自圣路易斯华盛顿大学生物化学和分子生物学系和神经疾病希望中心的杰出导师团队将监测和评估候选人的研究进展和职业目标的实现情况。作为职业发展计划的一部分，候选人将积极参加位于圣路易斯华盛顿大学的神经疾病希望中心和蛋白质聚集与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工作，并增加候选人研究的翻译潜力。

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.