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: 10610165
• 负责人: Yonghao Yu
• 金额: $ 32.23万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 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/10610165
• 关键词: ADP ribosylation; Acylation; Address; Amino Acids; Amyotrophic Lateral Sclerosis; Animal Model; Binding; Binding Proteins; Biological; Biological Process; Brain; Cell Death; Cell Nucleus; Cells; Cellular Stress; Cerebellar Ataxia; Cessation of life; Chemicals; DNA; DNA Damage; DNA Repair; Data; Diffuse; Enzymes; FDA approved; Family; Genetic; Genotoxic Stress; Heterogeneous-Nuclear Ribonucleoproteins; Human; Imidazole; In Vitro; Length; Malignant Neoplasms; Malignant neoplasm of ovary; Mass Spectrum Analysis; Mediating; Membrane; Messenger RNA; Methods; Modification; Molecular Conformation; Mutate; Nature; 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; Sampling; Seeds; Series; Side; Signal Transduction; Site; Stimulant; Structure; Surface; System; Traumatic Brain Injury; Work; acyl group; aspartylglutamate; base; biological adaptation to stress; biophysical tools; brain dysfunction; density; experience; experimental study; genotoxicity; inhibitor; malignant breast neoplasm; mouse model; nervous system disorder; 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. The PARylation level in a quiescent cell is usually very low. 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 neurological disorders (e.g., Parkinson’s disease, amyotrophic lateral sclerosis, traumatic brain injury and cerebellar ataxia). PARP1 is directly activated by a variety of neurotoxic stimulants, 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 develope 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 (Aim 1). Then we will use tunable footprinting to study how PARylation regulates phase-transition in vitro (Aim 2) and in intact nuclei (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 neurological disorders.

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，PARP1 是一种与细胞应激反应密切相关的核蛋白。静止细胞中的 PARylation 水平为 通常很低。为了应对基因毒性应激，PARP1 与带切口的 DNA 结合并迅速激活， 导致大量 PARylated 蛋白的合成并启动 DNA 损伤修复 （DDR）机制。事实上，FDA 最近已批准四种 PARP1 抑制剂用于治疗 BRCA- 突变的卵巢癌和/或乳腺癌。除了在人类环境中调节 DDR 的作用之外 在恶性肿瘤中，最近的证据表明 PARylation 是神经元中的死亡信号。重要的是， PARP1 的基因缺失或药物抑制可对大脑功能障碍提供深远的保护 许多神经系统疾病的动物模型（例如帕金森病、肌萎缩侧索硬化症、 创伤性脑损伤和小脑性共济失调）。 PARP1被多种神经毒性兴奋剂直接激活， 异常的 PARylation 会促进生物分子缩合物的形成。尽管既定的角色 PARylation 在相变调节中的作用，该过程的结构方面是难以捉摸的。致地址 为此，我们将利用我们已发表的工作和我实验室的丰富经验。这些初步数据是 主要集中在两个不同的程序上。首先，PARylation 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。我们最近能够克服这些挑战，并且 开发一种大规模质谱方法来全面表征 Asp- 和 Glu-PARylated 蛋白质组。使用这种方法，我们定义了各种不同条件下的全局 PARylated 蛋白质组。 遗传毒性条件。其次，生物分子凝聚体是一类无膜细胞器，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 开发了一种基于质谱的化学“足迹”方法，用于这些物质的结构分析 蛋白质原纤维。基于这些结果，我们将开发一种新颖的、可调节的足迹方法 生物分子凝聚体的结构动力学表征（目标 1）。然后我们将使用可调 足迹法研究 PARylation 如何在体外（目标 2）和完整细胞核（目标 3）中调节相变。这 从这些研究中获得的信息将提供对这一关键生物学的基本理解 过程，为靶向 PARP1 治疗神经系统疾病铺平了道路。