Role of ADP-Ribosylation in Stress Granules-Equipment Supplement

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修复灶和 用于制造核糖体亚基的核仁）。它们的形成、大小、数量和 然而，监管的动态仍不清楚。紧急研究表明，它们的形成可以部分地 这可以用一种被称为液-液相分离的生物物理现象来解释， 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 当蛋白质结合到一个共同的支架，如核酸DNA和RNA时， 蛋白质的缩合形成更高级的结构。我们之前发现，一个未被研究的 一种叫做聚腺苷二磷酸核糖（PAR）的核酸对于一类无膜细胞器的形成至关重要 与癌症、病毒感染和神经变性有关，称为应激颗粒。应力颗粒是 细胞质RNA-蛋白质组装体响应于应激物如缺氧而形成不同大小， 氧化应激和热休克。大多数颗粒成分与周围环境动态交换 细胞质，并且单个颗粒通过融合随着时间的推移而在尺寸上生长。值得注意的是，模型中的应力颗粒 神经病理性疾病如肌萎缩侧索硬化症（ALS）具有较慢的交换动力学， 更不容易融合然而，控制胁迫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。在本建议中，我们将（1）确定 PAR如何利用创新技术调节应力颗粒中的相分离，以确定关键的 ADP-核糖基化在细胞和体外形成应激颗粒的参数，和（2）确定是否 PAR-蛋白质相互作用利用诱变、活细胞成像、生物物理学和免疫学调节应激颗粒融合 方法和蛋白质组学。预计的影响：除了其在应力颗粒中的作用外，PAR对 形成时间和位置特异性的无膜结构，包括DNA修复复合物， 核仁。因此，该建议将通过定义生理相关的关键参数来推进该领域。 PAR介导的相分离，并通过鉴定这些现象所需的ADP-核糖基化蛋白。 鉴于PARP是可药物化的，并被制药公司积极瞄准， 抑制剂可以被设计成调节生理和病理性的代谢产物的形成和动力学。 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.