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

项目摘要 Poly-ADP-核糖化是一种蛋白质翻译后修饰(PTM)，由一种 称为聚腺苷二磷酸核糖聚合酶(PARP)的酶家族。在各种PARP酶中，PARP1 是一种核蛋白，与细胞应激反应密切相关。作为对基因毒性应激的反应，PARP1 与镍DNA结合并被迅速激活，导致合成大量的PARylated蛋白质 以及启动DNA损伤修复(DDR)机制。事实上，四种PARP1抑制剂最近已经被 FDA批准用于治疗BRCA突变的卵巢癌和/或乳腺癌。除了在调节方面的作用 在人类恶性肿瘤的背景下，最近的证据表明，PAR化是一种死亡信号 在神经元中。重要的是，PARP1的基因缺失或药物抑制提供了深刻的保护 在许多神经退行性疾病的动物模型中对抗脑功能障碍，包括阿尔茨海默氏症 疾病、帕金森氏病、肌萎缩侧索硬化症/肌萎缩侧索硬化症和额颞叶痴呆/FTD。PARP1是 由多种神经毒性刺激物(例如，病理性蛋白聚集体)直接激活，以及异常 对位作用促进生物分子缩合物的形成。尽管对偶联反应的作用已经确立 相变的规则，这一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我的实验室的丰富经验。这些初步数据主要是 专注于两个不同的项目。首先，对质谱学家来说，对偶联反应是一种臭名昭著的PTM，因为 它的易变性和异质性。我们最近能够克服这些挑战，并开发出一种大型- 天冬氨酸和谷氨酰化的标度质谱学综合表征 蛋白质组。利用这种方法，我们已经定义了不同遗传毒性下的全球PAR化蛋白质组 条件。第二，生物分子缩合物是一类无膜细胞器，其结构 动力学不太容易受到传统生物物理工具的影响。为了解决这个问题，我们之前开发了一个质量 基于光谱的化学“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 与肌萎缩侧索硬化症和肌萎缩侧索硬化症相关的生物分子凝聚物动力学(目标1)。然后我们将使用可调参数 足迹研究PAR化如何在体外调节相变(目标2)。最后，我们将使用可调参数 足迹法研究诱导多能干细胞来源的PAR化介导的相变 C9orf72介导的ALS和FTD的神经元(IPSN)和苍蝇模型(目标3)。从以下渠道获得的信息 这些研究将提供对这一关键生物过程的基本理解，为 以PARP1为靶点治疗ALS和FTD，以及更广泛的阿尔茨海默病相关痴呆。

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.