Role of ADP-ribosylation in Stress Granules

项目摘要 无膜结构在细胞中普遍存在，并执行独特的功能(例如，DNA 修复病灶和核仁以形成核糖体亚基)。他们通过的机制 然而，形成、大小、数量和动态是受调控的，但仍不清楚。紧急情况 研究表明，它们的形成可以部分地用生物物理现象来解释。 被称为液-液相分离，即核质和细胞质 被认为是像油和水一样稳定分离的复杂流体。相分离通常是 当蛋白质结合到常见的支架上时，如DNA和RNA， 从而导致蛋白质的缩合形成更高级别的结构。我们之前 发现一种被称为聚(ADP-核糖)(PAR)的未被研究的核酸对 一类无膜细胞器的形成与癌症、病毒感染和 神经退行性变称为应激颗粒。应激颗粒是细胞质的核糖核酸蛋白 对低氧、氧化应激等应激源的响应形成不同大小的组件 和中暑。大多数颗粒组件与周围环境动态交换 细胞质，随着时间的推移，单个颗粒通过融合而变大。值得注意的是，压力 神经病理疾病模型中的颗粒，如肌萎缩侧索硬化症(ALS)， 交换动力较慢，融合能力较差。然而，分子因素 控制应力颗粒的动态和融合(影响颗粒的大小和数量) 人们对此仍然知之甚少。在本提案中，我们将(1)确定PAR如何调节阶段 在应力颗粒中使用创新技术来确定ADP的关键参数 核糖化在细胞内和体外应激颗粒形成中的作用，以及(2)决定PAR-1是否在细胞内和体外形成应激颗粒。 蛋白质相互作用通过突变、活细胞成像、 生物物理学方法和蛋白质组学。预计影响：除了在压力颗粒中的作用外，PAR 对于形成时间和位置特定的无膜结构也是至关重要的， 包括DNA修复复合体和核仁。因此，这项提议将使这一领域向前推进 定义生理上相关的PAR介导的相分离的关键参数，并通过 鉴定这些现象所需的ADP核糖化蛋白。鉴于Parp是 下一代抑制剂是可药物的，并被制药公司积极瞄准，可能 旨在调节生理和病理的形成和动态 神经系统或其他疾病中的无膜结构。

Project Summary Membrane-less structures are prevalent in cells and executing unique functions (e.g., DNA repair foci and nucleoli for making ribosomal subunits). The mechanisms by which their formation, size, number, and dynamics are regulated, however, remain unclear. Emergent studies revealed that their formation can be partly explained by a biophysical phenomenon known as liquid-liquid phase separation, whereby the nucleoplasm and cytoplasm are considered complex fluids that stably segregate like oil and water. Phase separation is often triggered when proteins bind to a common scaffold such as the nucleic acids DNA and RNA, resulting in the condensation of proteins to form higher-order structures. We previously discovered that an under-studied nucleic acid called poly(ADP-ribose) (PAR) is critical for the formation of a class of membrane-less organelles implicated in cancer, virus infection and neurodegeneration called stress granules. Stress granules are cytoplasmic RNA-protein assemblies formed in different sizes in response to stressors such as hypoxia, oxidative stress and heat shock. Most granule components dynamically exchange with the surrounding cytoplasm, and individual granules grow in size over time through fusion. Notably, stress granules in models of neuropathological diseases, such as amyotrophic lateral sclerosis (ALS), have slower exchange dynamics and are less able to fuse. However, the molecular factors that control the stress granule dynamics and fusion (which affects size and number of granules) remain poorly understood. In this proposal, we will (1) determine how PAR regulates phase separation in stress granules using innovative techniques to define critical parameters of ADP- ribosylation for stress granule formation in cells and in vitro, and (2) determine whether PAR- protein interactions regulate stress granule fusion using mutagenesis, live-cell imaging, biophysical methods and proteomics. Projected Impact: Besides its role in stress granules, PAR is also critical for the formation of time- and location-specific membrane-less structures, including DNA repair complexes and nucleoli. This proposal will thus advance the field by defining critical parameters for physiologically relevant PAR-mediated phase separation and by identifying ADP-ribosylated proteins required for these phenomena. Given that PARPs are druggable and actively targeted by pharmaceutical companies, next-generation inhibitors may be designed to modulate the formation and dynamics of physiological and pathological membrane-less structures in neurological or other diseases.