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

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。静止细胞中的PAR化水平是 通常很低。在对遗传毒性应激的反应中，PARP 1与切口DNA结合并被迅速激活， 导致大量PAR化蛋白质的合成和DNA损伤修复的启动 (DDR)机制等事实上，FDA最近批准了四种PARP 1抑制剂用于治疗BRCA， 突变的卵巢癌和/或乳腺癌。除了在人的安全方面管理复员方案的作用外， 在恶性肿瘤中，最近的证据表明PAR化充当神经元中的死亡信号。重要的是， PARP 1的基因缺失或药理学抑制提供了对脑功能障碍的深刻保护， 许多神经疾病的动物模型（例如，帕金森氏症，肌萎缩侧索硬化症， 创伤性脑损伤和小脑共济失调）。PARP 1被多种神经毒性刺激物直接激活， 异常PAR化促进生物分子缩合物的形成。尽管已经确立了 PAR基化在调控相变过程中的结构方面是难以捉摸的。解决 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项目上。首先，PARylation是质谱学家的一个臭名昭著的PTM， 因为它的不稳定性和异质性。我们最近能够克服这些挑战， 开发一种大规模的质谱方法，以全面表征天冬氨酸和 Glu-PAR化蛋白质组。使用这种方法，我们已经定义了各种条件下的全局PAR化蛋白质组。 遗传毒性条件。其次，生物分子凝聚物是一类无膜细胞器， 结构动力学不太适合于传统的生物物理工具。为了解决这个问题，我们以前 开发了一种基于质谱的化学“足迹”方法，用于这些化合物的结构分析。 蛋白质纤维基于这些结果，我们将开发一种新的，可调的足迹方法， 生物分子凝聚物的结构动力学表征（目的1）。然后我们将使用可调 足迹法来研究PAR化如何在体外（Aim 2）和完整核（Aim 3）中调节相变。的 从这些研究中获得的信息将提供对这种关键生物学的基本理解。 这一过程为靶向PARP 1治疗神经系统疾病铺平了道路。

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.