Role of ADP-Ribosylation in Stress Granules-Equipment Supplement

项目总结 无膜结构在细胞中普遍存在，并执行独特的功能(例如，DNA修复焦点和 用于制造核糖体亚基的核仁)。它们的形成、大小、数量和 然而，动态受到监管，目前仍不清楚。新出现的研究表明，它们的形成可以部分地 用一种称为液-液相分离的生物物理现象来解释，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触发，如DNA和RNA，导致 蛋白质的缩合以形成更高级别的结构。我们之前发现，一个研究不足的人 核酸称为聚(adp-核糖)(Par)是形成一类无膜细胞器的关键。 与癌症、病毒感染和神经退行性变有关，称为应激颗粒。应激颗粒是 细胞质RNA-蛋白质组合形成不同大小的组合，以响应诸如低氧等应激因素， 氧化应激和热休克。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，应激颗粒在模型中 神经病理疾病，如肌萎缩侧索硬化症(ALS)，交换动力学和 更不能融合。然而，控制应力颗粒动力学和融合的分子因素 (影响颗粒的大小和数量)仍然知之甚少。在本提案中，我们将(1)确定 PAR如何使用创新技术调节应力颗粒中的相分离以定义关键 ADP-核糖化在细胞和体外应激颗粒形成中的参数，以及(2)确定 PAR-蛋白质相互作用通过突变、活细胞成像、生物物理 方法和蛋白质组学。预期影响：除了在压力颗粒中的作用外，PAR对 形成时间和位置特定的无膜结构，包括DNA修复复合体和 核仁。因此，这一提议将通过定义与生理相关的关键参数来推进场 PAR介导的相分离和鉴定这些现象所需的ADP核糖化蛋白。 鉴于PAP是可下药的，并积极成为制药公司的目标，下一代 抑制剂可以被设计成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.