Role of ADP-ribosylation in Stress Granules

ADP-核糖基化在应激颗粒中的作用

• 批准号: 10388732
• 负责人: Anthony K L Leung
• 金额: $ 25万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388732
• 关键词: ADP ribosylation; Affect; Amyotrophic Lateral Sclerosis; Binding Proteins; Biophysics; Cells; Complex; Cytoplasm; Cytoplasmic Granules; DNA; DNA Repair; Disease; Enzymes; Family; Heat-Shock Response; Higher Order Chromatin Structure; Hypoxia; In Vitro; Individual; Liquid substance; Location; Malignant Neoplasms; Mediating; Membrane; Modeling; Molecular; Mutagenesis; Nerve Degeneration; Neurologic; Nucleic Acids; Nucleoplasm; Oils; Organelles; Oxidative Stress; Pathologic; Pharmacologic Substance; Phase; Physical condensation; Physiological; Poly Adenosine Diphosphate Ribose; Proteins; Proteomics; RNA; Regulation; Repair Complex; Ribosomes; Role; Structure; Techniques; Therapeutic; Time; Virus Diseases; Water; biophysical techniques; design; inhibitor/antagonist; innovation; live cell imaging; next generation; response; scaffold; stress granule; stressor

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.

项目概要 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、数量和分布的机制 然而，动态受到调节仍不清楚。新兴研究表明，它们的形成可能部分是由于 这是通过一种称为液-液相分离的生物物理现象来解释的，其中核质 细胞质和细胞质被认为是像油和水一样稳定分离的复杂流体。相分离经常发生 当蛋白质与核酸 DNA 和 RNA 等共同支架结合时触发，从而产生 蛋白质缩合形成高阶结构。我们之前发现，一个未被充分研究的 称为聚（ADP-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、病毒感染和神经退行性变有关的称为应激颗粒。应力颗粒是 细胞质 RNA-蛋白质组装体以不同大小形成，以响应缺氧、 氧化应激和热休克。大多数颗粒成分与周围环境动态交换 细胞质和单个颗粒通过融合随着时间的推移而尺寸增大。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)，交换动力学较慢， 融合能力较差。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 如何使用创新技术来调节应力颗粒中的相分离来定义关键 细胞内和体外应激颗粒形成的 ADP-核糖基化参数，以及 (2) 确定是否 PAR-蛋白质相互作用利用诱变、活细胞成像、生物物理调节应激颗粒融合 方法和蛋白质组学。预计影响：除了在应激颗粒中的作用外，PAR 对于 形成时间和位置特异性的无膜结构，包括 DNA 修复复合物和 核仁。因此，该提案将通过定义生理相关的关键参数来推进该领域的发展 PAR 介导的相分离并通过识别这些现象所需的 ADP 核糖基化蛋白。 鉴于 PARP 具有可成药性，并且是制药公司的积极目标，下一代 抑制剂可以被设计来调节生理和病理的形成和动态 神经或其他疾病中的无膜结构。