A Chemical Footprinting Approach towards Poly-ADP-Ribosylation-regulated Biomolecular Condensation

聚 ADP 核糖基化调节生物分子缩合的化学足迹方法

• 批准号: 10389853
• 负责人: Yonghao Yu
• 金额: $ 8.87万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10389853
• 关键词: ADP ribosylation; Acylation; Address; Alzheimer' s Disease; Alzheimer' s disease related dementia; Amino Acids; Amyotrophic Lateral Sclerosis; Animal Model; Benchmarking; Binding; Binding Proteins; Biological; Biological Process; C9ORF72; Cell Death; Cell Nucleus; Cells; Cellular Stress; Cessation of life; Chemicals; DNA; DNA Damage; DNA Repair; Data; Defect; Diffuse; Electron Microscopy; Enzymes; FDA approved; Family; Frontotemporal Dementia; Genes; Genetic; Genotoxic Stress; Heterogeneous-Nuclear Ribonucleoproteins; Human; Imidazole; In Vitro; Induced pluripotent stem cell derived neurons; Length; Malignant Neoplasms; Malignant neoplasm of ovary; Mass Spectrum Analysis; Mediating; Membrane; Messenger RNA; Methods; Modeling; Modification; Molecular Conformation; Mutate; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotoxins; Nitrogen; Nuclear; Nuclear Pore; Nuclear Protein; Nucleic Acids; Organelles; Oxygen; Parkinson Disease; Pathogenesis; Pathologic; Pathway interactions; Pharmacology; Phase Transition; Physical condensation; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymers; Post-Translational Protein Processing; Process; Protein Analysis; Proteins; Proteome; Publishing; RNA; Reagent; Recombinants; Regulation; Role; Seeds; Series; Side; Signal Transduction; Site; Stimulant; Stress; Structure; Surface; Translations; Work; amyotrophic lateral sclerosis therapy; aspartylglutamate; base; biological adaptation to stress; biophysical techniques; biophysical tools; brain dysfunction; density; experience; fly; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genotoxicity; inhibitor; malignant breast neoplasm; neuron loss; neurotoxic; novel; programs; protein aggregation; response; self assembly; small molecule; spatiotemporal

Project Summary Poly-ADP-ribosylation (PARylation) is a protein posttranslational modification (PTM) that is catalyzed by a family of enzymes called Poly-ADP-ribose polymerases (PARPs). Among the various PARP enzymes, PARP1 is a nuclear protein that is critically involved in cell stress responses. In response to genotoxic stress, PARP1 binds to nicked DNA and is rapidly activated, resulting in the synthesis of a large number of PARylated proteins and initiation of the DNA damage repair (DDR) mechanisms. Indeed, four PARP1 inhibitors have recently been approved by the FDA to treat BRCA-mutated ovarian and/or breast cancers. Besides the role in regulating DDR in the context of human malignancies, recent evidence suggests that PARylation serves as a death signal in neurons. Importantly, genetic deletion or pharmacological inhibition of PARP1 offers profound protection against brain dysfunction in the animal models of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis/ALS and frontotemporal dementia/FTD. PARP1 is directly activated by a variety of neurotoxic stimulants (e.g., pathologic protein aggregates), and aberrant PARylation promotes the formation of biomolecular condensates. Despite the established role of PARylation in the regulation of phase-transition, the structural aspects of this process are elusive. To address this, we will leverage our published work and the extensive experience of my lab. These preliminary data are largely focused on two different programs. First, PARylation is a notorious PTM for mass spectrometrists, because of its labile and heterogenous nature. We recently were able to overcome these challenges, and develop a large- scale mass spectrometric approach towards comprehensive characterization of the Asp- and Glu-PARylated proteome. Using this approach, we have defined the global PARylated proteome under various genotoxic conditions. Second, biomolecular condensates are a class of membrane-less organelles, whose structural dynamics are less amenable to traditional biophysical tools. To address this, we previously developed a mass spectrometry-based chemical “footprinting” method for the structural analysis of these protein fibrils. Based on these results, we will develop a novel, tunable footprinting approach for the characterization of the structural dynamics of biomolecular condensates that are relevant to ALS and FTD (Aim 1). Then we will use tunable footprinting to study how PARylation regulates phase-transition in vitro (Aim 2). Finally, we will use tunable footprinting to characterize PARylation-mediated phase-transition in induced pluripotent stem-cell-derived neurons (iPSN) and fly models of C9orf72-mediated ALS and FTD (Aim 3). The information garnered from these studies will provide a fundamental understanding of this critical biological process, paving the way for targeting PARP1 for the treatment of ALS and FTD, and more broadly, Alzheimer's disease related dementias.

项目摘要 多聚ADP核糖基化（PAR化）是一种蛋白质翻译后修饰（PTM）， 聚ADP核糖聚合酶（PARP）。在各种PARP酶中，PARP 1 是一种与细胞应激反应密切相关的核蛋白。在遗传毒性应激反应中，PARP 1 与带切口的DNA结合并迅速活化，导致大量PAR化蛋白质的合成 和启动DNA损伤修复（DDR）机制。事实上，最近已经有四种PARP 1抑制剂 FDA批准用于治疗BRCA突变的卵巢癌和/或乳腺癌。除了调节 在人类恶性肿瘤的背景下，最近的证据表明，PAR化作为死亡信号 在神经元中。重要的是，PARP 1的基因缺失或药理学抑制提供了深刻的保护 在包括阿尔茨海默氏症在内的许多神经退行性疾病的动物模型中对抗脑功能障碍 疾病、帕金森病、肌萎缩侧索硬化症/ALS和额颞叶痴呆/FTD。PARP 1是 由多种神经毒性刺激物直接激活（例如，病理性蛋白质聚集体），以及异常的 PAR化促进生物分子缩合物的形成。尽管PARylation的作用已经确立， 相变的调节，这个过程的结构方面是难以捉摸的。为了解决这个问题，我们将 利用我们发表的成果和我实验室的丰富经验。这些初步数据主要是 专注于两个不同的项目。首先，PAR化对于质谱学家来说是一个臭名昭著的PTM，因为 它的不稳定和异质性。我们最近克服了这些挑战，并开发了一个大型的- 规模质谱方法对全面表征的天冬氨酸和谷氨酸PAR 蛋白质组使用这种方法，我们已经定义了各种遗传毒性下的全局PAR化蛋白质组， 条件其次，生物分子凝聚物是一类无膜细胞器，其结构 动力学不太适合传统的生物物理工具。为了解决这个问题，我们以前开发了一个质量 基于光谱学的化学“足迹”方法用于这些蛋白质原纤维的结构分析。基于 这些结果，我们将开发一种新的，可调的足迹法的表征结构 与ALS和FTD相关的生物分子缩合物的动力学（目标1）。然后我们将使用可调 足迹法来研究PAR化如何在体外调节相变（目的2）。最后，我们将使用可调 足迹法表征PAR化介导的诱导多能干细胞衍生的 神经元（iPSN）和C9 orf 72介导的ALS和FTD的苍蝇模型（Aim 3）。信息收集自 这些研究将为这一关键的生物学过程提供基本的理解， 靶向PARP 1治疗ALS和FTD，更广泛地说，治疗阿尔茨海默病相关痴呆。