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

基本信息

批准号：
10524783
负责人：
Yonghao Yu
金额：
$ 41.13万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10524783
关键词：
ADP ribosylation Acetylation Acylation Address Alzheimer&apos s Disease Alzheimer&apos 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 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 Phase Transition Physical condensation Poly Adenosine Diphosphate Ribose Poly(ADP-ribose) Polymerases Polymers Post-Translational Protein Processing Process Protein Analysis Proteins Proteome Publishing RNA Reaction Reagent Recombinants Regulation Role Series Side Signal Transduction Site Stimulant Stress Structure Surface Translations Work amyotrophic lateral sclerosis therapy 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 pharmacologic programs protein aggregation response self assembly small molecule spatiotemporal

项目摘要

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.

项目概要聚 ADP 核糖基化 (PARylation) 是一种蛋白质翻译后修饰 (PTM)，由称为聚 ADP 核糖聚合酶 (PARP) 的酶家族在各种 PARP 酶中，有 PARP1。 PARP1 是一种核蛋白，在细胞应激反应中发挥重要作用。与带切口的 DNA 结合并迅速激活，导致大量 PARylated 蛋白的合成事实上，最近有四种 PARP1 抑制剂被开发出来。除具有调节作用外，还获得 FDA 批准用于治疗 BRCA 突变的卵巢癌和/或乳腺癌。 DDR 在人类恶性肿瘤的背景下，最近的证据表明 PARylation 可作为死亡信号重要的是，PARP1 的基因缺失或药理学抑制提供了深远的保护。在许多神经退行性疾病（包括阿尔茨海默病）的动物模型中对抗脑功能障碍病、帕金森病、肌萎缩侧索硬化症/ALS 和额颞叶痴呆/PARP1。直接被各种神经毒性兴奋剂（例如病理性蛋白质聚集体）激活，并且异常尽管 PARylation 在生物分子缩合物中的作用已被证实，但 PARylation 仍可促进生物分子缩合物的形成。相变的调控，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。这些初步数据主要是利用我们已发表的工作和我实验室的丰富经验。首先，PARylation 对于质谱学家来说是一个臭名昭著的 PTM，因为我们最近能够克服这些挑战，并开发出一个大型的。规模质谱方法全面表征 Asp- 和 Glu-PARylated 使用这种方法，我们定义了各种基因毒性下的全局 PARylated 蛋白质组。其次，生物分子缩合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，我们之前开发了一个质量。基于光谱测定的化学“足迹”方法对这些蛋白质原纤维进行结构分析。根据这些结果，我们将开发一种新颖的、可调节的足迹方法来表征结构与 ALS 和 FTD 相关的生物分子凝聚体动力学（目标 1）。足迹法研究 PARylation 如何在体外调节相变（目标 2）。足迹法表征诱导多能干细胞衍生的 PARylation 介导的相变神经元 (iPSN) 和 C9orf72 介导的 ALS 和 FTD 果蝇模型（目标 3）。这些研究将为这一关键的生物过程提供基本的了解，为靶向 PARP1 来治疗 ALS 和 FTD，更广泛地说，治疗阿尔茨海默病相关的痴呆症。