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

TDP-43 磷酸化在蛋白质功能和神经变性中的作用

基本信息

批准号：
8620730
负责人：
Yuna Ayala
金额：
$ 20.57万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-15 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8620730
关键词：
Achievement Address Affect Agreement Alzheimer&apos s Disease Amino Acids Amyotrophic Lateral Sclerosis Award Biochemistry Cell Culture Techniques Cellular Assay Commit Cytoplasmic Granules Cytoplasmic Inclusion DNA-Binding Proteins Data Defect Dementia Development Diagnosis Disease Embryo Event Faculty Frontotemporal Lobar Degenerations Future Goals Homeostasis Inclusion Bodies Investigation Knockout Mice Light Link Lobar Location Maps Mass Spectrum Analysis Mediating Mentors Modeling Modification Molecular Molecular Biology Monitor Mutate Mutation National Institute of Neurological Disorders and Stroke Nerve Degeneration Neurodegenerative Disorders Neurons Neurosciences Research Nuclear Nuclear Protein Parkinson Disease Pathogenesis Pathogenicity Pathology Pathway interactions Patients Phosphorylation Phosphorylation Site Physiological Positioning Attribute Post-Translational Protein Processing Preparation Process Proteins Proteome RNA RNA Processing RNA Splicing RNA-Binding Proteins Regulation Reporting Research Role Saints Serine Site Solubility Stress Structure Testing Training Universities Washington base career career development interest mutant nervous system disorder novel novel diagnostics novel therapeutics programs protein TDP-43 protein aggregation protein function public health relevance response stressor tool trafficking translational study

项目摘要

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.

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 是在肌萎缩侧索硬化症 (ALS) 和前颞叶变性 (FTLD) 这两种主要神经变性形式的细胞质包涵体中积累的主要疾病蛋白。 TDP-43 主要在细胞核中介导 RNA 加工。 TDP-43 聚集伴随着核定位的丧失，这表明该蛋白质至少一种核功能的缺乏与神经变性有关。此外，在家族性和散发性 ALS-FTLD 中发现的 40 多种显性突变凸显了 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磷酸位点。根据它们的位置，报告和预测的磷酸位点可能会对关键的 TDP-43 过程产生深远的影响，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 突变可能会增加蛋白质磷酸化。我们假设 TDP-43 受磷酸化调节，并且一些与疾病相关的突变改变了磷酸化谱，对蛋白质功能产生有害后果。我们建议（目标 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确定的新型磷酸位点；（目标 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；（目标 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位点。将在成熟的细胞测定中使用定点突变体来探讨磷酸化对蛋白质功能的作用，以监测 TDP-43 活性和溶解度。目前缺乏有关 TDP-43 生理磷酸化以及与患者衍生突变体相关的信息，这凸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 的因素和细胞途径，并有助于阐明导致神经退行性变的过程。该候选人对介导 RNA 加工的蛋白质的表征有着长期的兴趣，特别是 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，并重点关注神经系统疾病机制。作为圣路易斯大学爱德华·A·多伊西生物化学和分子生物学系最近成立的独立教员，该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以及希望神经疾病中心的优秀导师团队将监督和评估研究进展以及候选人职业目标的实现情况。作为职业发展计划的一部分，候选人将积极参与圣路易斯华盛顿大学希望神经疾病中心和希望中心蛋白质聚集和神经变性项目。这将极大地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。