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.

项目摘要多-ADP-核糖基化（Parylation）是一种蛋白质后修饰（PTM），由A催化酶家族称为多ADP-核糖聚合酶（PARPS）。在各种PARP酶中，PARP1 是一种与细胞应激反应有关的核蛋白。响应于遗传毒性应激，PARP1 结合到划痕的DNA并迅速激活，导致大量荷蛋白的合成 DNA损伤修复（DDR）机制的主动性。确实，最近有四个PARP1抑制剂经FDA批准以治疗BRCA突变的卵巢和/或乳腺癌。除了确定的角色 DDR在人类恶性肿瘤的背景下，最近的证据表明，pary被用作死亡信号在神经元中。重要的是，PARP1的遗传缺失或药物抑制提供了深刻的保护反对许多神经退行性疾病的动物模型中的脑功能障碍，包括阿尔茨海默氏症疾病，帕金森氏病，肌萎缩性侧硬化/ALS和额颞痴呆/FTD。 Parp1是直接被多种神经毒性刺激剂（例如病理蛋白聚集体）激活，并且异常荷载促进了生物分子冷凝物的形成。尽管Parylation在相变的调节，该过程的结构方面是难以捉摸的。为了解决这个问题，我们将利用我们已发表的工作和实验室的丰富经验。这些初步数据在很大程度上是专注于两个不同的程序。首先，由于质谱师的臭名昭著的PTM，因为它不稳定和异质性。我们最近能够克服这些挑战，并发展了一个大型尺度质谱法，以全面表征ASP和Glu-Parylated 使用这种方法，我们在各种遗传毒素下定义了全局的蛋白质组状况。其次，生物分子冷凝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，我们以前开发了质量基于光谱法的化学“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，我们将开发一种新颖的，可调的足迹方法来表征结构与ALS和FTD相关的生物分子冷凝物的动力学（AIM 1）。然后我们将使用可调的足迹研究如何在体外调节相位转变（AIM 2）。最后，我们将使用可调足迹以表征在诱导的多能干细胞衍生的诱导多能中的Parylation介导的相变神经元（IPSN）和C9ORF72介导的ALS和FTD的飞行模型（AIM 3）。从中获得的信息这些研究将为这一关键的生物学过程提供基本的理解，为靶向PARP1治疗ALS和FTD，更广泛地是阿尔茨海默氏病有关的痴呆症。